Calcified Tissue International

, Volume 78, Issue 5, pp 302–313 | Cite as

Effects of Radiation Therapy on Chondrocytes In Vitro

  • B. S. MarguliesEmail author
  • J. A. Horton
  • Y. Wang
  • T. A. Damron
  • M. J. Allen


The negative irradiation complications of growth loss leading to limb length asymmetry and pathological fracture incurred following radiation therapy in pediatric patients has led to a renewed interest in understanding the specific effects of irradiation on the growth plate and the surrounding bone. In the present report, we examined the radiation therapy effects on primary rat growth cartilage chondrocytes in order to determine the chondrocyte radiosensitivity relative to other bone cell constituents and tumor cells, the postirradiation temporal progression of radiation-induced alterations in chondrocyte function, and the time course for the functional restoration of chondrocyte pathways that drive the eventual recovery in growth function. We employed an in vitro primary rat costochondral growth cartilage cell culture model system to evaluate the radiation therapy effects on proliferative chondrocytes using serial radiation doses (0–20 Gy) that are well within the clinically relevant range. Following irradiation, all of the following occurred in a dose-dependent manner: proliferation decreased, cytotoxicity increased, several markers of apoptosis increased, markers of radiation-induced cellular differentiation increased, and cell synthetic activity was disturbed. Alterations in proliferation, cell death, and induction of apoptosis are likely due to a transient radiation-induced derangement of the parathyroid hormone-related protein-Indian hedgehog proliferation-maturation pathway. Alterations in cellular differentiation and cell synthetic activity are novel observations for chondrocytes. Further, these results correspond very well to our previous work in an in vivo Sprague-Dawley rat model, making this model particularly relevant to researching the radiation therapy effects on longitudinal growth.


Chondrocyte Cell growth Growth factor Cell differentiation Apoptosis 



This work was supported by NIH NCCAM F31 predoctoral fellowship (AT01994) and NIH NCI 2R01 (CA083892).


  1. 1.
    Butler MS, Robertson WW Jr, Rate W, D’Angio GJ, Drummond DS (1990) Skeletal sequelae of radiation therapy for malignant childhood tumors. Clin Orthop 251:235–240PubMedGoogle Scholar
  2. 2.
    Merchant TE, Kushner BH, Sheldon JM, LaQuaglia M, Healey JH (1999) Effect of low-dose radiation therapy when combined with surgical resection for Ewing sarcoma. Med Pediatr Oncol 33:65–70CrossRefPubMedGoogle Scholar
  3. 3.
    Koscielniak E, Morgan M, Treuner J (2002) Soft tissue sarcoma in children: prognosis and management. Paediatr Drugs 4:21–28PubMedGoogle Scholar
  4. 4.
    Wall JE, Kaste SC, Greenwald CA, Jenkins JJ, Douglass EC, Pratt CB (1996) Fractures in children treated with radiotherapy for soft tissue sarcoma. Orthopedics 19:657–64PubMedGoogle Scholar
  5. 5.
    Damron TA, Margulies B, Biskup D, Spadaro JA (2001) Amifostine before fractionated irradiation protects bone growth in rats better than fractionation alone. Int J Radiat Oncol Biol Phys 50:479–483CrossRefPubMedGoogle Scholar
  6. 6.
    Pateder DB, Eliseev RA, O’Keefe RJ, Schwarz EM, Okunieff P, Constine LS, Puzas JE, Rosier RN (2001) The role of autocrine growth factors in radiation damage to the epiphyseal growth plate. Radiat Res 155:847–857PubMedGoogle Scholar
  7. 7.
    Pateder DB, Sheu TJ, O’Keefe RJ, Puzas JE, Schwarz EM, Constine LS, Okunieff P, Rosier RN (2002) Role of pentoxifylline in preventing radiation damage to epiphyseal growth plate chondrocytes. Radiat Res 157:62–68PubMedGoogle Scholar
  8. 8.
    Damron TA, Margulies B, Strauss J, Spadaro J, Farnum CE (2003) Sequential histomorphometric changes following irradiation of the growth plate with and without radioprotectant. J Bone Joint Surg Am 85:1302–1313PubMedGoogle Scholar
  9. 9.
    Margulies B, Morgan H, Allen M, Strauss J, Spadaro J, Damron T (2003) Increased bone density following irradiation and the radioprotectant drug amifostine in a rat model. Am J Clin Oncol 26:e106–e114CrossRefPubMedGoogle Scholar
  10. 10.
    Cruz-Orive LM, Weibel ER (1990) Recent stereological methods for cell biology: a brief survey. Am J Physiol 258:148–156Google Scholar
  11. 11.
    Roederer M, Treister A, Moore W, Herzenberg LA (2001) Probability binning comparison: a metric for quantitating univariate distribution differences. Cytometry 45:37–46CrossRefPubMedGoogle Scholar
  12. 12.
    Margulies B, Damron T, Allen M (2003) Differential effects of radiation and radioprotectant on Ewing’s sarcoma, rhabdomyosarcoma, bone marrow cells and osteoblasts. Trans Orthopedic Research Society Meeting, Abstract 229, New Orleans, LA, February 2003Google Scholar
  13. 13.
    Ruchon AF, Marcinkiewicz M, Ellefsen K, Basaka, Aubin J, Crine P, Boileau G (2000) Cellular Localization of neprilysin in mouse bone tissue and putative role in hydrolysis of osteogenic peptides. J Bone Miner Res 24:1266–1274CrossRefGoogle Scholar
  14. 14.
    Enomoto H, Enomoto-Iwamoto M, Iwamoto M, Nomura S, Himeno M, Kitamura Y, Kishimoto T, Komori T (2000) Cbfa1 is a positive regulatory factor in chondrocyte maturation. J Biol Chem 275:8695–8702CrossRefPubMedGoogle Scholar
  15. 15.
    Hiranuma H, Jikko A, Iwamoto M, Fuchihata H (1996) Effects of X-ray irradiation on terminal differentiation and cartilage matrix calcification of rabbit growth plate chondrocytes in culture. Bone 18:233–238CrossRefPubMedGoogle Scholar
  16. 16.
    Hofbauer KH, Gess B, Lohaus C, Meyer HE, Katschinski D, Kurtz A (2003) Oxygen tension regulates the expression of a group of procollagen hydroxylases. Eur J Biochem 270:4515–4522CrossRefPubMedGoogle Scholar
  17. 17.
    Damron TA, Mathur S, Horton JA, Strauss J, Margulies B, Grant W, Farnum CE, Spadaro JA (2004) Temporal changes in PTHrP, Bcl-2, Bax, caspase, TGF-beta, and FGF-2 expression following growth plate irradiation with or without radioprotectant. J Histochem Cytochem 52:157–167PubMedGoogle Scholar
  18. 18.
    Beier F (2005) Cell-cycle control and the cartilage growth plate. J Cell Physiol 202:1–8CrossRefPubMedGoogle Scholar
  19. 19.
    Alvarez J, Sohn P, Zeng X, Doetschman T, Robbins DJ, Serra R (2002) TGFβ2 mediates the effects of Hedgehog on hypertrophic differentiation and PTHrP expression. Development 129:1913–1924PubMedGoogle Scholar
  20. 20.
    Kabayashi T, Chung U, Schipani E, Starbuck M, Karsenty G, Katagiri T, Goad DL, Lanske B, Kronenberg HM (2002) PTHrP and indian hedgehog control differentiation of growth plate chondrocytes at multiple steps. Development 129:2977–2986Google Scholar
  21. 21.
    Weksler NB, Lunstrum GP, Reid ES, Horton WA (1999) Differential effects of fibroblast growth factor (FGF) 9 and FGF2 on proliferation, differentiation and terminal differentiation of chondrocytic cells in vitro. Biochem J 342:677–682CrossRefPubMedGoogle Scholar
  22. 22.
    Trippel SB, Wroblewski J, Makower AM, Whelan MC, Schoenfeld D, Doctrow SR (1993) Regulation of growth-plate chondrocytes by insulin-like growth-factor I and basic fibroblastic growth factor. J Bone Joint Surg Am 75:177–189PubMedGoogle Scholar
  23. 23.
    Olney RC, Wang J, Sylvester JE, Mougey EB (2004) Growth factor regulation of human growth plate chondrocytes proliferation in vitro. Biochem Biophys Res Commun 317:1171–1182CrossRefPubMedGoogle Scholar
  24. 24.
    Krejci P, Bryja V, Pachernik J, Hampl A, Pogue R, Mekikian P, Wilcox WR (2004) FGF2 inhibits proliferation and alters the cartilage-like phenotype of RCS cells. Exp Cell Res 297:152–164CrossRefPubMedGoogle Scholar
  25. 25.
    Naski MC, Wang JX, Ornitz DM (1996) Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nat Genet 13:233–237CrossRefPubMedGoogle Scholar
  26. 26.
    Laplantine E, Rossi F, Sahni M, Basilico C, Cobrinik D (2002) FGF signaling targets the pRb-related p107 and p130 proteins to induce chondrocyte growth arrest. J Cell Biol 158:741–750CrossRefPubMedGoogle Scholar
  27. 27.
    Erez N, Stambolsky P, Shats I, Milyavsky M, Kachko T, Rotter V (2004) Hypoxia-dependent regulation of PHD1: cloning and characterization of the human PHD1/EGLN2 gene promoter. FEBS Lett 567:311–315CrossRefPubMedGoogle Scholar
  28. 28.
    Gustafsson E, Aszodi A, Ortega N, Hunziker EB, Denker HW, Werb Z, Fassler R (2003) Role of Collagen II and perlecan in skeletal development. Ann Ny Acad Sci 995:140–150PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • B. S. Margulies
    • 1
  • J. A. Horton
    • 1
  • Y. Wang
    • 1
  • T. A. Damron
    • 1
  • M. J. Allen
    • 1
  1. 1.Department of Orthopedic SurgeryState University of New York, Upstate Medical UniversitySyracuseUSA

Personalised recommendations