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Delayed Fracture Healing in Growth Differentiation Factor 5-deficient Mice: A Pilot Study

  • Basic Research
  • Published:
Clinical Orthopaedics and Related Research®

Abstract

Background

Growth differentiation factor-5 (GDF-5) is a key regulator of skeletogenesis and bone repair and induces bone formation in spinal fusions and nonunion applications by enhancing chondrocytic and osteocytic differentiation and stimulating angiogenesis. Elucidating the contribution of GDF-5 to fracture repair may support its clinical application in complex fractures.

Questions/purpose

We therefore asked whether the absence of GDF-5 during fracture repair impaired bone healing as assessed radiographically, histologically, and mechanically.

Methods

In this pilot study, we performed tibial osteotomies on 10-week-old male mice, stabilized by intramedullary and extramedullary nailing. Healing was assessed radiographically and histologically on Days 1 (n = 1 wild-type; n = 5 bp [brachopodism]), 5 (n = 3 wild-type; n = 3 bp), 10 (n = 6 wild-type; n = 3 bp), 14 (n = 6 wild-type; n = 6 bp), 21 (n = 6 wild-type; n = 6 bp), 28 (n = 7 wild-type; n = 6 bp), and 56 (n = 6 wild-type; n = 6 bp) after fracture. After 10 (n = 7 wild-type; n = 7 bp contralateral and n = 3 bp fractured tibiae), 14 (n = 6 wild-type; n = 6 bp), 21 (n = 6 wild-type; n = 6 bp), 28 (n = 6 wild-type; n = 3 bp), and 56 (n = 8 wild-type; n = 6 bp) days, the callus cross-sectional area was calculated. We characterized the mechanical integrity of the healing fracture by yield stress and Young’s modulus at 28 (n = 6 wild-type; n = 3 bp) and 56 (n = 8 wild-type; n = 6 bp) days postfracture.

Results

The absence of GDF-5 impaired cartilaginous matrix deposition in the callus and reduced callus cross-sectional area. After 56 days, the repaired bp fracture was mechanically comparable to that of controls.

Conclusions

Although GDF-5 deficiency did not compromise long-term fracture healing, a delay in cartilage formation and remodeling supports roles for GDF-5 in the early phase of bone repair.

Clinical Relevance

Local delivery of GDF-5 to clinically difficult fractures may simulate cartilage formation in the callus and support subsequent remodeling.

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References

  1. Al-Yahyaee SA, Al-Kindi MN, Habbal O, Kumar DS. Clinical and molecular analysis of Grebe acromesomelic dysplasia in an Omani family. Am J Med Genet A. 2003;121A:9–14.

    Article  PubMed  Google Scholar 

  2. Bai X, Xiao Z, Pan Y, Hu J, Pohl J, Wen J, Li L. Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes. Biochem Biophys Res Commun. 2004;325:453–460.

    Article  PubMed  CAS  Google Scholar 

  3. Chang SC, Hoang B, Thomas JT, Vukicevic S, Luyten FP, Ryba NJ, Kozak CA, Reddi AH, Moos M Jr. Cartilage-derived morphogenetic proteins: new members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development. J Biol Chem. 1994;269:28227–28234.

    PubMed  CAS  Google Scholar 

  4. Cheung KM, Kaluarachi K, Andrew G, Lu W, Chan D, Cheah KS. An externally fixed femoral fracture model for mice. J Orthop Res. 2003;21:685–690.

    Article  PubMed  Google Scholar 

  5. Chhabra A, Zijerdi D, Zhang J, Kline A, Balian G, Hurwitz S. BMP-14 deficiency inhibits long bone fracture healing: a biochemical, histologic, and radiographic assessment. J Orthop Trauma. 2005;19:629–634.

    Article  PubMed  Google Scholar 

  6. Cho TJ, Gerstenfeld LC, Einhorn TA. Differential temporal expression of members of the transforming growth factor beta superfamily during murine fracture healing. J Bone Miner Res. 2002;17:513–520.

    Article  PubMed  CAS  Google Scholar 

  7. Coleman CM, Loredo GA, Lo CW, Tuan RS. Correlation of GDF5 and connexin 43 mRNA expression during embryonic development. Anat Rec A Discov Mol Cell Evol Biol. 2003;275:1117–1121.

    Article  PubMed  Google Scholar 

  8. Coleman CM, Tuan RS. Functional role of growth/differentiation factor 5 in chondrogenesis of limb mesenchymal cells. Mech Dev. 2003;120:823–836.

    Article  PubMed  CAS  Google Scholar 

  9. Coleman CM, Tuan RS. Growth/differentiation factor 5 enhances chondrocyte maturation. Dev Dyn. 2003;228:208–216.

    Article  PubMed  CAS  Google Scholar 

  10. Costa T, Ramsby G, Cassia F, Peters KR, Soares J, Correa J, Quelce-Salgado A, Tsipouras P. Grebe syndrome: clinical and radiographic findings in affected individuals and heterozygous carriers. Am J Med Genet. 1998;75:523–529.

    Article  PubMed  CAS  Google Scholar 

  11. Erlacher L, McCartney J, Piek E, ten Dijke P, Yanagishita M, Oppermann H, Luyten FP. Cartilage-derived morphogenetic proteins and osteogenic protein-1 differentially regulate osteogenesis. J Bone Miner Res. 1998;13:383–392.

    Article  PubMed  CAS  Google Scholar 

  12. Everman DB, Bartels CF, Yang Y, Yanamandra N, Goodman FR, Mendoza-Londono JR, Savarirayan R, White SM, Graham JM Jr, Gale RP, Svarch E, Newman WG, Kleckers AR, Francomano CA, Govindaiah V, Singh L, Morrison S, Thomas JT, Warman ML. The mutational spectrum of brachydactyly type C. Am J Med Genet. 2002;112:291–296.

    Article  PubMed  Google Scholar 

  13. Faiyaz-Ul-Haque M, Ahmad W, Wahab A, Haque S, Azim AC, Zaidi SH, Teebi AS, Ahmad M, Cohn DH, Siddique T, Tsui LC. Frameshift mutation in the cartilage-derived morphogenetic protein 1 (CDMP1) gene and severe acromesomelic chondrodysplasia resembling Grebe-type chondrodysplasia. Am J Med Genet. 2002;111:31–37.

    Article  PubMed  Google Scholar 

  14. Faiyaz-Ul-Haque M, Ahmad W, Zaidi SH, Haque S, Teebi AS, Ahmad M, Cohn DH, Tsui LC. Mutation in the cartilage-derived morphogenetic protein-1 (CDMP1) gene in a kindred affected with fibular hypoplasia and complex brachydactyly (DuPan syndrome). Clin Genet. 2002;61:454–458.

    Article  PubMed  CAS  Google Scholar 

  15. Francis-West PH, Abdelfattah A, Chen P, Allen C, Parish J, Ladher R, Allen S, MacPherson S, Luyten FP, Archer CW. Mechanisms of GDF-5 action during skeletal development. Development. 1999;126:1305–1315.

    PubMed  CAS  Google Scholar 

  16. Galjaard RJ, van der Ham LI, Posch NA, Dijkstra PF, Oostra BA, Hovius SE, Timmenga EJ, Sonneveld GJ, Hoogeboom AJ, Heutink P. Differences in complexity of isolated brachydactyly type C cannot be attributed to locus heterogeneity alone. Am J Med Genet. 2001;98:256–262.

    Article  PubMed  CAS  Google Scholar 

  17. Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. 2003;88:873–884.

    Article  PubMed  CAS  Google Scholar 

  18. Gruber R, Mayer C, Bobacz K, Krauth MT, Graninger W, Luyten FP, Erlacher L. Effects of cartilage-derived morphogenetic proteins and osteogenic protein-1 on osteochondrogenic differentiation of periosteum-derived cells. Endocrinology. 2001;142:2087–2094.

    Article  PubMed  CAS  Google Scholar 

  19. Gruber R, Mayer C, Schulz W, Graninger W, Peterlik M, Watzek G, Luyten FP, Erlacher L. Stimulatory effects of cartilage-derived morphogenetic proteins 1 and 2 on osteogenic differentiation of bone marrow stromal cells. Cytokine. 2000;12:1630–1638.

    Article  PubMed  CAS  Google Scholar 

  20. Gruneberg H, Lee AJ. The anatomy and development of brachypodism in the mouse. J Embryol Exp Morphol. 1973;30:119–141.

    PubMed  CAS  Google Scholar 

  21. Hatakeyama Y, Tuan RS, Shum L. Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis. J Cell Biochem. 2004;91:1204–1217.

    Article  PubMed  CAS  Google Scholar 

  22. Hiltunen A, Vuorio E, Aro HT. A standardized experimental fracture in the mouse tibia. J Orthop Res. 1993;11:305–312.

    Article  PubMed  CAS  Google Scholar 

  23. Histing T, Garcia P, Matthys R, Leidinger M, Holstein JH, Kristen A, Pohlemann T, Menger MD. An internal locking plate to study intramembranous bone healing in a mouse femur fracture model. J Orthop Res. 2010;28:397–402.

    PubMed  Google Scholar 

  24. Holder-Espinasse M, Escande F, Mayrargue E, Dieux-Coeslier A, Fron D, Doual-Bisser A, Boute-Benejean O, Robert Y, Porchet N, Manouvrier-Hanu S. Angel shaped phalangeal dysplasia, hip dysplasia, and positional teeth abnormalities are part of the brachydactyly C spectrum associated with CDMP-1 mutations. J Med Genet. 2004;41:e78.

    Article  PubMed  CAS  Google Scholar 

  25. Hotten G, Neidhardt H, Jacobowsky B, Pohl J. Cloning and expression of recombinant human growth/differentiation factor 5. Biochem Biophys Res Commun. 1994;204:646–652.

    Article  PubMed  CAS  Google Scholar 

  26. Jahng TA, Fu TS, Cunningham BW, Dmitriev AE, Kim DH. Endoscopic instrumented posterolateral lumbar fusion with Healos and recombinant human growth/differentiation factor-5. Neurosurgery. 2004;54:171–180; discussion 180–181.

    Article  PubMed  Google Scholar 

  27. Kjaer KW, Eiberg H, Hansen L, van der Hagen CB, Rosendahl K, Tommerup N, Mundlos S. A mutation in the receptor binding site of GDF5 causes Mohr-Wriedt brachydactyly type A2. J Med Genet. 2006;43:225–231.

    Article  PubMed  CAS  Google Scholar 

  28. Koch H, Jadlowiec JA, Fu FH, Nonn J, Merk HR, Hollinger JO, Campbell PG. [The effect of growth/differentiation factor-5 (GDF-5) on genotype and phenotype in human adult mesenchymal stem cells] [in German]. Z Orthop Ihre Grenzgeb. 2004;142:248–253.

    Article  PubMed  CAS  Google Scholar 

  29. Kuniyasu H, Hirose Y, Ochi M, Yajima A, Sakaguchi K, Murata M, Pohl J. Bone augmentation using rhGDF-5-collagen composite. Clin Oral Implants Res. 2003;14:490–499.

    Article  PubMed  Google Scholar 

  30. Landauer W. Brachypodism: a recessive mutation of house-mice. J Hered. 1952;43:724–732.

    Google Scholar 

  31. Lin K, Thomas JT, McBride OW, Luyten FP. Assignment of a new TGF-beta superfamily member, human cartilage-derived morphogenetic protein-1, to chromosome 20q11.2. Genomics. 1996;34:150–151.

    Article  PubMed  CAS  Google Scholar 

  32. Mikic B, Battaglia TC, Taylor EA, Clark RT. The effect of growth/differentiation factor-5 deficiency on femoral composition and mechanical behavior in mice. Bone. 2002;30:733–737.

    Article  PubMed  CAS  Google Scholar 

  33. Polinkovsky A, Robin NH, Thomas JT, Irons M, Lynn A, Goodman FR, Reardon W, Kant SG, Brunner HG, van der Burgt I, Chitayat D, McGaughran J, Donnai D, Luyten FP, Warman ML. Mutations in CDMP1 cause autosomal dominant brachydactyly type C. Nat Genet. 1997;17:18–19.

    Article  PubMed  CAS  Google Scholar 

  34. Sato M, Ochi T, Nakase T, Hirota S, Kitamura Y, Nomura S, Yasui N. Mechanical tension-stress induces expression of bone morphogenetic protein (BMP)-2 and BMP-4, but not BMP-6, BMP-7, and GDF-5 mRNA, during distraction osteogenesis. J Bone Miner Res. 1999;14:1084–1095.

    Article  PubMed  CAS  Google Scholar 

  35. Schwabe GC, Turkmen S, Leschik G, Palanduz S, Stover B, Goecke TO, Mundlos S. Brachydactyly type C caused by a homozygous missense mutation in the prodomain of CDMP1. Am J Med Genet A. 2004;124A:356–363.

    Article  PubMed  Google Scholar 

  36. Seemann P, Schwappacher R, Kjaer KW, Krakow D, Lehmann K, Dawson K, Stricker S, Pohl J, Ploger F, Staub E, Nickel J, Sebald W, Knaus P, Mundlos S. Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2. J Clin Invest. 2005;115:2373–2381.

    Article  PubMed  CAS  Google Scholar 

  37. Sena K, Sumner DR, Virdi AS. Modulation of VEGF expression in rat bone marrow stromal cells by GDF-5. Connect Tissue Res. 2007;48:324–331.

    Article  PubMed  CAS  Google Scholar 

  38. Shen FH, Zeng Q, Lv Q, Choi L, Balian G, Li X, Laurencin CT. Osteogenic differentiation of adipose-derived stromal cells treated with GDF-5 cultured on a novel three-dimensional sintered microsphere matrix. Spine J. 2006;6:615–623.

    Article  PubMed  Google Scholar 

  39. Spiro RC, Liu L, Heidaran MA, Thompson AY, Ng CK, Pohl J, Poser JW. Inductive activity of recombinant human growth and differentiation factor-5. Biochem Soc Trans. 2000;28:362–368.

    Article  PubMed  CAS  Google Scholar 

  40. Spiro RC, Thompson AY, Poser JW. Spinal fusion with recombinant human growth and differentiation factor-5 combined with a mineralized collagen matrix. Anat Rec. 2001;263:388–395.

    Article  PubMed  CAS  Google Scholar 

  41. Stelzer C, Winterpacht A, Spranger J, Zabel B. Grebe dysplasia and the spectrum of CDMP1 mutations. Pediatr Pathol Mol Med. 2003;22:77–85.

    Article  PubMed  CAS  Google Scholar 

  42. Storm EE, Huynh TV, Copeland NG, Jenkins NA, Kingsley DM, Lee SJ. Limb alterations in brachypodism mice due to mutations in a new member of the TGF beta-superfamily. Nature. 1994;368:639–643.

    Article  PubMed  CAS  Google Scholar 

  43. Storm EE, Kingsley DM. GDF5 coordinates bone and joint formation during digit development. Dev Biol. 1999;209:11–27.

    Article  PubMed  CAS  Google Scholar 

  44. Szczaluba K, Hilbert K, Obersztyn E, Zabel B, Mazurczak T, Kozlowski K. Du Pan syndrome phenotype caused by heterozygous pathogenic mutations in CDMP1 gene. Am J Med Genet A. 2005;138:379–383.

    PubMed  CAS  Google Scholar 

  45. Thomas JT, Kilpatrick MW, Lin K, Erlacher L, Lembessis P, Costa T, Tsipouras P, Luyten FP. Disruption of human limb morphogenesis by a dominant negative mutation in CDMP1. Nat Genet. 1997;17:58–64.

    Article  PubMed  CAS  Google Scholar 

  46. Tsumaki N, Tanaka K, Arikawa-Hirasawa E, Nakase T, Kimura T, Thomas JT, Ochi T, Luyten FP, Yamada Y. Role of CDMP-1 in skeletal morphogenesis: promotion of mesenchymal cell recruitment and chondrocyte differentiation. J Cell Biol. 1999;144:161–173.

    Article  PubMed  CAS  Google Scholar 

  47. Wolfman NM, Hattersley G, Cox K, Celeste AJ, Nelson R, Yamaji N, Dube JL, DiBlasio-Smith E, Nove J, Song JJ, Wozney JM, Rosen V. Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family. J Clin Invest. 1997;100:321–330.

    Article  PubMed  CAS  Google Scholar 

  48. Yamashita H, Shimizu A, Kato M, Nishitoh H, Ichijo H, Hanyu A, Morita I, Kimura M, Makishima F, Miyazono K. Growth/differentiation factor-5 induces angiogenesis in vivo. Exp Cell Res. 1997;235:218–226.

    Article  PubMed  CAS  Google Scholar 

  49. Yeh LC, Tsai AD, Lee JC. Cartilage-derived morphogenetic proteins induce osteogenic gene expression in the C2C12 mesenchymal cell line. J Cell Biochem. 2005;95:173–188.

    Article  PubMed  CAS  Google Scholar 

  50. Yoshimoto T, Yamamoto M, Kadomatsu H, Sakoda K, Yonamine Y, Izumi Y. Recombinant human growth/differentiation factor-5 (rhGDF-5) induced bone formation in murine calvariae. J Periodontal Res. 2006;41:140–147.

    Article  PubMed  CAS  Google Scholar 

  51. Zeng Q, Li X, Beck G, Balian G, Shen FH. Growth and differentiation factor-5 (GDF-5) stimulates osteogenic differentiation and increases vascular endothelial growth factor (VEGF) levels in fat-derived stromal cells in vitro. Bone. 2007;40:374–381.

    Article  PubMed  CAS  Google Scholar 

  52. Zeng Q, Li X, Choi L, Beck G, Balian G, Shen FH. Recombinant growth/differentiation factor-5 stimulates osteogenic differentiation of fat-derived stromal cells in vitro. Connect Tissue Res. 2006;47:264–270.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

We thank Erika Bickler for her technical contribution to this work.

Author information

Authors and Affiliations

  1. Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Service, Bethesda, MD, USA

    Cynthia M. Coleman PhD, Brooke H. Scheremeta DO, Amanda T. Boyce PhD, Robert L. Mauck PhD & Rocky S. Tuan PhD

  2. Regenerative Medicine Institute, National University of Ireland Galway, Galway City, County Galway, Ireland

    Cynthia M. Coleman PhD

  3. Schneider Children’s Hospital, New Hyde Park, NY, USA

    Brooke H. Scheremeta DO

  4. McKay Orthopaedic Research Laboratory, Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA, USA

    Robert L. Mauck PhD

  5. Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 221, Pittsburgh, PA, 15219, USA

    Rocky S. Tuan PhD

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  1. Cynthia M. Coleman PhD
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  2. Brooke H. Scheremeta DO
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  3. Amanda T. Boyce PhD
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  4. Robert L. Mauck PhD
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  5. Rocky S. Tuan PhD
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Corresponding author

Correspondence to Rocky S. Tuan PhD.

Additional information

One or more of the authors (RT) received funding from National Institutes of Health grant ZO1 AR41131.

Each author certifies that his or her institution approved the animal protocol for this investigation and that all investigations were conducted in conformity with ethical principles of research.

This work was performed at the Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Service, Bethesda, MD, USA.

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Coleman, C.M., Scheremeta, B.H., Boyce, A.T. et al. Delayed Fracture Healing in Growth Differentiation Factor 5-deficient Mice: A Pilot Study. Clin Orthop Relat Res 469, 2915–2924 (2011). https://doi.org/10.1007/s11999-011-1912-0

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  • DOI: https://doi.org/10.1007/s11999-011-1912-0

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