Advertisement

Journal of Molecular Medicine

, Volume 91, Issue 5, pp 625–636 | Cite as

Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo

  • Magali CucchiariniEmail author
  • Patrick Orth
  • Henning Madry
Original Article

Abstract

Direct gene transfer strategies are of promising value to treat articular cartilage defects. Here, we tested the ability of a recombinant adeno-associated virus (rAAV) SOX9 vector to enhance the repair of cartilage lesions in vivo. The candidate construct was provided to osteochondral defects in rabbit knee joints vis-à-vis control (lacZ) vector treatment and to cells relevant of the repair tissue (mesenchymal stem cells, chondrocytes). Efficient, long-term transgene expression was noted within the lesions (up to 16 weeks) and in cells in vitro (21 days). Administration of the SOX9 vector was capable of stimulating the biological activities in vitro and over time in vivo. SOX9 treatment in vivo was well tolerated, leading to improved cartilage repair processes with enhanced production of major matrix components. Remarkably, application of rAAV SOX9 delayed premature terminal differentiation and hypertrophy in the newly formed cartilage, possible due to contrasting effects of SOX9 on RUNX2 and β-catenin osteogenic expression in this area. Most strikingly, SOX9 treatment improved the reconstitution of the subchondral bone in the defects, possibly due to an increase in RUNX2 expression in this location. These findings show the potential of direct rAAV gene delivery as an efficient tool to treat cartilage lesions.

Keywords

Articular cartilage defects Rabbits Gene transfer rAAV SOX9 

Notes

Acknowledgments

This study was supported by the German Research Society (DFG CU 55/1-1,/1-2,/1-3) and the German Osteoarthritis Foundation (Deutsche Arthrose-Hilfe). The authors declare no competing financial or other interests. We thank R. J. Samulski (The Gene Therapy Center, University of North Carolina, Chapel Hill, NC) and 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 G. Scherer (Institute for Human Genetics and Anthropology, Albert-Ludwig University, Freiburg, Germany) for the human SOX9 cDNA. We are also thankful to T. Thurn and G. Schmitt (Center of Experimental Orthopaedics, Homburg/Saar, Germany) for technical assistance and to D. Zurakowski (Children’s Hospital, Orthopaedic Surgery and Biostatistics, Harvard Medical School, Boston, MA, USA), M. D. Menger (Institute for Experimental Surgery, Homburg/Saar, Germany), D. Kohn (Department of Orthopaedic Surgery, Homburg/Saar, Germany), and A. Pinzano (CNRS, UMR 7561, Vandoeuvre-lès-Nancy, France) for helpful discussions.

Conflict of interest

The authors declare no conflict of interest.

References

  1. 1.
    Abe T, Yamada H, Nakajima H, Kikuchi T, Takaishi H, Tadakuma T, Fujikawa K, Toyama Y (2003) Repair of full-thickness cartilage defects using liposomal transforming growth factor-beta1. J Orthop Sci 8:92–101PubMedCrossRefGoogle Scholar
  2. 2.
    Menendez MI, Clark DJ, Carlton M, Flanigan DC, Jia G, Sammet S, Weisbrode SE, Knopp MV, Bertone AL (2011) Direct delayed human adenoviral BMP-2 or BMP-6 gene therapy for bone and cartilage regeneration in a pony osteochondral model. Osteoarthr Cartil 19:1066–1075PubMedCrossRefGoogle Scholar
  3. 3.
    Cucchiarini M, Madry H, Ma C, Thurn T, Zurakowski D, Menger MD, Kohn D, Trippel SB, Terwilliger EF (2005) Improved tissue repair in articular cartilage defects in vivo by rAAV-mediated overexpression of human fibroblast growth factor 2. Mol Ther 12:229–238PubMedCrossRefGoogle Scholar
  4. 4.
    Nixon AJ, Fortier LA, Williams J, Mohammed H (1999) Enhanced repair of extensive articular defects by insulin-like growth factor-I-laden fibrin composites. J Orthop Res 17:475–487PubMedCrossRefGoogle Scholar
  5. 5.
    Morisset S, Frisbie DD, Robbins PD, Nixon AJ, McIlwraith CW (2007) IL-1ra/IGF-1 gene therapy modulates repair of microfractured chondral defects. Clin Orthop Relat Res 462:221–228PubMedCrossRefGoogle Scholar
  6. 6.
    Klinger P, Surmann-Schmitt C, Brem M, Swoboda B, Distler JH, Carl HD, von der Mark K, Hennig FF, Gelse K (2011) Chondromodulin 1 stabilizes the chondrocyte phenotype and inhibits endochondral ossification of porcine cartilage repair tissue. Arthritis Rheum 63:2721–2731PubMedCrossRefGoogle Scholar
  7. 7.
    Cao L, Yang F, Liu G, Yu D, Li H, Fan Q, Gan Y, Tang T, Dai K (2011) The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells. Biomaterials 32:3910–3920PubMedCrossRefGoogle Scholar
  8. 8.
    Ikeda T, Kamekura S, Mabuchi A, Kou I, Seki S, Takato T, Nakamura K, Kawaguchi H, Ikegawa S, Chung UI (2004) The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage. Arthritis Rheum 50:3561–3573PubMedCrossRefGoogle Scholar
  9. 9.
    Kupcsik L, Stoddart MJ, Li Z, Benneker LM, Alini M (2010) Improving chondrogenesis: potential and limitations of SOX9 gene transfer and mechanical stimulation for cartilage tissue engineering. Tissue Eng A 16:1845–1855CrossRefGoogle Scholar
  10. 10.
    Venkatesan JK, Ekici M, Madry H, Schmitt G, Kohn D, Cucchiarini M (2012) SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells. Stem Cell Res Ther 3:22–36PubMedCrossRefGoogle Scholar
  11. 11.
    Cucchiarini M, Thurn T, Weimer A, Kohn D, Terwilliger EF, Madry H (2007) Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9. Arthritis Rheum 56:158–167PubMedCrossRefGoogle Scholar
  12. 12.
    Li Y, Tew SR, Russell AM, Gonzalez KR, Hardingham TE, Hawkins RE (2004) Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9. Tissue Eng 10:575–584PubMedCrossRefGoogle Scholar
  13. 13.
    Cucchiarini M, Ekici M, Schetting S, Kohn D, Madry H (2011) Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2. Tissue Eng A 17:1921–1933CrossRefGoogle Scholar
  14. 14.
    Pagnotto MR, Wang Z, Karpie JC, Ferretti M, Xiao X, Chu CR (2007) Adeno-associated viral gene transfer of transforming growth factor-beta1 to human mesenchymal stem cells improves cartilage repair. Gene Ther 14:804–813PubMedCrossRefGoogle Scholar
  15. 15.
    Stender S, Murphy M, O’Brien T, Stengaard C, Ulrich-Vinther M, Soballe K, Barry F (2007) Adeno-associated viral vector transduction of human mesenchymal stem cells. Eur Cell Mater 13:93–99PubMedGoogle Scholar
  16. 16.
    Samulski RJ, Chang LS, Shenk T (1987) 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 61:3096–3101PubMedGoogle Scholar
  17. 17.
    Samulski RJ, Chang LS, Shenk T (1989) Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 63:3822–3828PubMedGoogle Scholar
  18. 18.
    Sellers RS, Peluso D, Morris EA (1997) The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage. J Bone Joint Surg Am 79:1452–1463PubMedGoogle Scholar
  19. 19.
    Orth P, Cucchiarini M, Kaul G, Ong MF, Graber S, Kohn DM, Madry H (2012) Temporal and spatial migration pattern of the subchondral bone plate in a rabbit osteochondral defect model. Osteoarthr Cartil 20:1161–1169PubMedCrossRefGoogle Scholar
  20. 20.
    Orth P, Kaul G, Cucchiarini M, Zurakowski D, Menger MD, Kohn D, Madry H (2011) Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo. Knee Surg Sports Traumatol Arthrosc 19:2119–2130PubMedCrossRefGoogle Scholar
  21. 21.
    Shapiro F, Koide S, Glimcher MJ (1993) Cell origin and differentiation in the repair of full-thickness defects of articular cartilage. J Bone Joint Surg Am 75:532–553PubMedGoogle Scholar
  22. 22.
    Wuelling M, Vortkamp A (2010) Transcriptional networks controlling chondrocyte proliferation and differentiation during endochondral ossification. Pediatr Nephrol 25:625–631PubMedCrossRefGoogle Scholar
  23. 23.
    Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM, Tabin CJ (1996) Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273:613–622PubMedCrossRefGoogle Scholar
  24. 24.
    Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C (2005) Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 8:727–738PubMedCrossRefGoogle Scholar
  25. 25.
    Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR et al (2004) Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev 18:1072–1087PubMedCrossRefGoogle Scholar
  26. 26.
    Evans CH, Liu FJ, Glatt V, Hoyland JA, Kirker-Head C, Walsh A, Betz O, Wells JW, Betz V, Porter RM et al (2009) Use of genetically modified muscle and fat grafts to repair defects in bone and cartilage. Eur Cell Mater 18:96–111PubMedGoogle Scholar
  27. 27.
    Katayama R, Wakitani S, Tsumaki N, Morita Y, Matsushita I, Gejo R, Kimura T (2004) Repair of articular cartilage defects in rabbits using CDMP1 gene-transfected autologous mesenchymal cells derived from bone marrow. Rheumatology 43:980–985PubMedCrossRefGoogle Scholar
  28. 28.
    Liu TM, Guo XM, Tan HS, Hui JH, Lim B, Lee EH (2011) Zinc-finger protein 145, acting as an upstream regulator of SOX9, improves the differentiation potential of human mesenchymal stem cells for cartilage regeneration and repair. Arthritis Rheum 63:2711–2720PubMedCrossRefGoogle Scholar
  29. 29.
    Madry H, Kaul G, Cucchiarini M, Stein U, Zurakowski D, Remberger K, Menger MD, Kohn D, Trippel SB (2005) Enhanced repair of articular cartilage defects in vivo by transplanted chondrocytes overexpressing insulin-like growth factor I (IGF-I). Gene Ther 12:1171–1179PubMedCrossRefGoogle Scholar
  30. 30.
    Mason JM, Breitbart AS, Barcia M, Porti D, Pergolizzi RG, Grande DA (2000) Cartilage and bone regeneration using gene-enhanced tissue engineering. Clin Orthop Relat Res 379:S171–S178PubMedCrossRefGoogle Scholar
  31. 31.
    Vogt S, Wexel G, Tischer T, Schillinger U, Ueblacker P, Wagner B, Hensler D, Wilisch J, Geis C, Wubbenhorst D et al (2009) The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects. Biomaterials 30:2385–2392PubMedCrossRefGoogle Scholar
  32. 32.
    Goldring MB, Fukuo K, Birkhead JR, Dudek E, Sandell LJ (1994) Transcriptional suppression by interleukin-1 and interferon-gamma of type II collagen gene expression in human chondrocytes. J Cell Biochem 54:85–99PubMedCrossRefGoogle Scholar
  33. 33.
    Anraku Y, Mizuta H, Sei A, Kudo S, Nakamura E, Senba K, Hiraki Y (2009) Analyses of early events during chondrogenic repair in rat full-thickness articular cartilage defects. J Bone Miner Metab 27:272–286PubMedCrossRefGoogle Scholar
  34. 34.
    Bi W, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B (1999) Sox9 is required for cartilage formation. Nat Genet 22:85–89PubMedCrossRefGoogle Scholar
  35. 35.
    Ng LJ, Wheatley S, Muscat GE, Conway-Campbell J, Bowles J, Wright E, Bell DM, Tam PP, Cheah KS, Koopman P (1997) SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse. Dev Biol 183:108–121PubMedCrossRefGoogle Scholar
  36. 36.
    Wright E, Hargrave MR, Christiansen J, Cooper L, Kun J, Evans T, Gangadharan U, Greenfield A, Koopman P (1995) The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos. Nat Genet 9:15–20PubMedCrossRefGoogle Scholar
  37. 37.
    Zhao Q, Eberspaecher H, Lefebvre V, De Crombrugghe B (1997) Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev Dyn 209:377–386PubMedCrossRefGoogle Scholar
  38. 38.
    Hattori T, Muller C, Gebhard S, Bauer E, Pausch F, Schlund B, Bosl MR, Hess A, Surmann-Schmitt C, von der Mark H et al (2010) SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development 137:901–911PubMedCrossRefGoogle Scholar
  39. 39.
    Zhou G, Zheng Q, Engin F, Munivez E, Chen Y, Sebald E, Krakow D, Lee B (2006) Dominance of SOX9 function over RUNX2 during skeletogenesis. Proc Natl Acad Sci U S A 103:19004–19009PubMedCrossRefGoogle Scholar
  40. 40.
    Topol L, Chen W, Song H, Day TF, Yang Y (2009) Sox9 inhibits Wnt signaling by promoting beta-catenin phosphorylation in the nucleus. J Biol Chem 284:3323–3333PubMedCrossRefGoogle Scholar
  41. 41.
    Guo X, Zheng Q, Yang S, Shao Z, Yuan Q, Pan Z, Tang S, Liu K, Quan D (2006) Repair of full-thickness articular cartilage defects by cultured mesenchymal stem cells transfected with the transforming growth factor beta1 gene. Biomed Mater 1:206–215PubMedCrossRefGoogle Scholar
  42. 42.
    Kuroda R, Usas A, Kubo S, Corsi K, Peng H, Rose T, Cummins J, Fu FH, Huard J (2006) Cartilage repair using bone morphogenetic protein 4 and muscle-derived stem cells. Arthritis Rheum 54:433–442PubMedCrossRefGoogle Scholar
  43. 43.
    Goodrich LR, Hidaka C, Robbins PD, Evans CH, Nixon AJ (2007) Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model. J Bone Joint Surg Br 89:672–685PubMedGoogle Scholar
  44. 44.
    Cucchiarini M, Terwilliger EF, Kohn D, Madry H (2009) Remodelling of human osteoarthritic cartilage by FGF-2, alone or combined with Sox9 via rAAV gene transfer. J Cell Mol Med 13:2476–2488PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Magali Cucchiarini
    • 1
    Email author
  • Patrick Orth
    • 1
    • 2
  • Henning Madry
    • 1
    • 2
  1. 1.Center of Experimental OrthopaedicsSaarland University Medical CenterHomburg/SaarGermany
  2. 2.Department of Orthopaedics and Orthopaedic SurgerySaarland University Medical CenterHomburg/SaarGermany

Personalised recommendations