, Volume 35, Issue 4, pp 1378–1391

Protection Against Titanium Particle-Induced Inflammatory Osteolysis by the Proteasome Inhibitor Bortezomib In Vivo

  • Xin Mao
  • Xiaoyun Pan
  • Song Zhao
  • Xiaochun Peng
  • Tao Cheng
  • Xianlong Zhang


Wear particle-induced vascularized granulomatous inflammation and subsequent inflammatory osteolysis is the most common cause of aseptic loosening after total joint replacement (TJR); however, the precise mechanism by which this occurs is unclear. This study investigates the effects of the proteasome inhibitor bortezomib (Bzb) on the expression of key biochemical markers of bone metabolism and vascularised granulomatous tissues, such as receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), vascular endothelial growth factor (VEGF) and tumor necrosis factor receptor-associated factor 6 (TRAF6). In addition, the effect of Bzb on apoptosis of CD68+ cells was examined. A total of 32 female BALB/C mice were randomly divided into four groups. After implantation of calvaria bone from syngeneic littermates, titanium (Ti) particles were injected into established air pouches for all mice (excluding negative controls) to provoke inflammatory osteolysis. Subsequently, Bzb was administered at a ratio of 0, 0.1, or 0.5 mg/kg on day 1, 4, 8, and 11 post-surgery to alleviate this response. All of the air pouches were harvested 14 days after the surgical procedure and were processed for molecular and histological analysis. The results demonstrated that Ti injection elevated the expression of RANKL, OPG, VEGF, and TRAF6 at both the gene and protein levels, increased counts of infiltrated cells and thickness of air pouch membranes, and elevated the apoptosis index (AI) of CD68+ cells. Bzb treatment significantly improved Ti particle-induced implanted bone osteolysis, attenuated vascularised granulomatous tissues and elevated AI of CD68+ cells. Therefore, the proteasome pathway may represent an effective therapeutic target for the prevention and treatment of aseptic loosening.


titanium aseptic loosening bortezomib osteolysis apoptosis 


  1. 1.
    Ingham, E., and J. Fisher. 2005. The role of macrophages in osteolysis of total joint replacement. Biomaterials 26(11): 1271–1286.PubMedCrossRefGoogle Scholar
  2. 2.
    Wang, M., P. Sharkey, and R. Tuan. 2004. Particle bioreactivity and wear-mediated osteolysis. The Journal of Arthroplasty 19(8): 1028–1038.PubMedCrossRefGoogle Scholar
  3. 3.
    Ren, W.P., D.C. Markel, R. Zhang, X. Peng, B. Wu, H. Monica, and P.H. Wooley. 2006. Association between UHMWPE particle-induced inflammatory osteoclastogenesis and expression of RANKL, VEGF, and Flt-1 in vivo. Biomaterials 27(30): 5161–5169.PubMedCrossRefGoogle Scholar
  4. 4.
    Al-Saffar, N., J. Mah, Y. Kadoya, and P.A. Revell. 1995. Neovascularisation and the induction of cell adhesion molecules in response to degradation products from orthopaedic implants. Annals of the Rheumatic Diseases 54(3): 201–208.PubMedCrossRefGoogle Scholar
  5. 5.
    Ren, W., R. Zhang, D.C. Markel, B. Wu, X. Peng, M. Hawkins, and P.H. Wooley. 2007. Blockade of vascular endothelial growth factor activity suppresses wear debris-induced inflammatory osteolysis. Journal of Rheumatology 34(1): 27–35.PubMedGoogle Scholar
  6. 6.
    Markel, D.C., R. Zhang, T. Shi, M. Hawkins, and W. Ren. 2009. Inhibitory effects of erythromycin on wear debris-induced VEGF/Flt-1 gene production and osteolysis. Inflammation Research 58(7): 413–421.CrossRefGoogle Scholar
  7. 7.
    Zhang, W., Peng, X., Cheng, T., and Zhang, X. 2011. Vascular endothelial growth factor gene silencing suppresses wear debris-induced inflammation. Int Orthop. Available from URL: (doi:10.1007/s00264-011-1252-4).
  8. 8.
    Anandarajah, A.P. 2009. Role of RANKL in bone diseases. Trends in Endocrinology and Metabolism 20(2): 88–94.PubMedCrossRefGoogle Scholar
  9. 9.
    Bord, S., D. Ireland, S. Beavan, and J. Compston. 2003. The effects of estrogen on osteoprotegerin, RANKL, and estrogen receptor expression in human osteoblasts. Bone 32(2): 136–141.PubMedCrossRefGoogle Scholar
  10. 10.
    Ye, H., J.R. Arron, B. Lamothe, M. Cirilli, T. Kobayashi, N.K. Shevde, D. Segal, O.K. Dzivenu, M. Vologodskaia, and M. Yim. 2002. Distinct molecular mechanism for initiating TRAF6 signalling. Nature 418(6896): 443–447.PubMedCrossRefGoogle Scholar
  11. 11.
    Armstrong, A.P., M.E. Tometsko, M. Glaccum, C.L. Sutherland, D. Cosman, and W.C. Dougall. 2002. A RANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function. Journal of Biological Chemistry 277(46): 44347–44356.PubMedCrossRefGoogle Scholar
  12. 12.
    Boyle, W.J., W.S. Simonet, and D.L. Lacey. 2003. Osteoclast differentiation and activation. Nature 423(6937): 337–342.PubMedCrossRefGoogle Scholar
  13. 13.
    Kobayashi, N., Y. Kadono, A. Naito, K. Matsumoto, T. Yamamoto, S. Tanaka, and J. Inoue. 2001. Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis. EMBO Journal 20(6): 1271–1280.PubMedCrossRefGoogle Scholar
  14. 14.
    Elliott, P.J., and J.S. Ross. 2001. The proteasome. American Journal of Clinical Pathology 116(5): 637–646.PubMedCrossRefGoogle Scholar
  15. 15.
    Wickner, S., M.R. Maurizi, and S. Gottesman. 1999. Posttranslational quality control: folding, refolding, and degrading proteins. Science 286(5446): 1888–1893.PubMedCrossRefGoogle Scholar
  16. 16.
    Richardson, P.G., B. Barlogie, J. Berenson, S. Singhal, S. Jagannath, D. Irwin, S.V. Rajkumar, G. Srkalovic, M. Alsina, and R. Alexanian. 2003. A phase 2 study of bortezomib in relapsed, refractory myeloma. The New England Journal of Medicine 348(26): 2609–2617.PubMedCrossRefGoogle Scholar
  17. 17.
    von Metzler, I., H. Krebbel, M. Hecht, R.A. Manz, C. Fleissner, M. Mieth, M. Kaiser, C. Jakob, J. Sterz, L. Kleeberg, U. Heider, and O. Sezer. 2007. Bortezomib inhibits human osteoclastogenesis. Leukemia 21(9): 2025–2034.CrossRefGoogle Scholar
  18. 18.
    Heider, U., M. Kaiser, C. Müller, C. Jakob, I. Zavrski, C.O. Schulz, C. Fleissner, M. Hecht, and O. Sezer. 2006. Bortezomib increases osteoblast activity in myeloma patients irrespective of response to treatment. European Journal of Haematology 77(3): 233–238.PubMedCrossRefGoogle Scholar
  19. 19.
    Arpinati, M., G. Chirumbolo, B. Nicolini, C. Agostinelli, and D. Rondelli. 2008. Selective apoptosis of monocytes and monocyte-derived DCs induced by bortezomib (Velcade). Bone Marrow Transplantation 43(3): 253–259.PubMedCrossRefGoogle Scholar
  20. 20.
    Rakshit, D.S., K. Ly, T.K. Sengupta, B.J. Nestor, T.P. Sculco, L.B. Ivashkiv, and P.E. Purdue. 2006. Wear debris inhibition of anti-osteoclastogenic signaling by interleukin-6 and interferon- gamma mechanistic insights and implications for periprosthetic osteolysis. The Journal of Bone and Joint Surgery American 88(4): 788–799.CrossRefGoogle Scholar
  21. 21.
    Wooley, P.H., R. Morren, J. Andary, S. Sud, S.Y. Yang, L. Mayton, D. Markel, A. Sieving, and S. Nasser. 2002. Inflammatory responses to orthopaedic biomaterials in the murine air pouch. Biomaterials 23(2): 517–526.PubMedCrossRefGoogle Scholar
  22. 22.
    Livak, K.J., and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4): 402–408.PubMedCrossRefGoogle Scholar
  23. 23.
    Ren, W., D.C. Markel, R. Schwendener, Y. Ding, B. Wu, and P.H. Wooley. 2008. Macrophage depletion diminishes implant-wear-induced inflammatory osteolysis in a mouse model. Journal of Biomedical Materials Research. Part A 85(4): 1043–1051.PubMedCrossRefGoogle Scholar
  24. 24.
    Purdue, P.E., P. Koulouvaris, B.J. Nestor, and T.P. Sculco. 2006. The central role of wear debris in periprosthetic osteolysis. HSS Journal 2(2): 102–113.PubMedCrossRefGoogle Scholar
  25. 25.
    Goodman, S.B., M. Trindade, T. Ma, M. Genovese, and R.L. Smith. 2005. Pharmacologic modulation of periprosthetic osteolysis. Clinical Orthopaedics and Related Research 430: 39–45.PubMedCrossRefGoogle Scholar
  26. 26.
    Ren, W., X.H. Li, B.D. Chen, and P.H. Wooley. 2004. Erythromycin inhibits wear debris-induced osteoclastogenesis by modulation of murine macrophage NF-κB activity. Journal of Orthopaedic Research 22(1): 21–29.PubMedCrossRefGoogle Scholar
  27. 27.
    Miyanishi, K., M.C.D. Trindade, T. Ma, S.B. Goodman, D.J. Schurman, and R.L. Smith. 2003. Periprosthetic osteolysis: induction of vascular endothelial growth factor from human monocyte/macrophages by orthopaedic biomaterial particles. Journal of Bone and Mineral Research 18(9): 1573–1583.PubMedCrossRefGoogle Scholar
  28. 28.
    Senger, D.R., S.J. Galli, A.M. Dvorak, C.A. Perruzzi, V.S. Harvey, and H.F. Dvorak. 1983. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219(4587): 983–985.PubMedCrossRefGoogle Scholar
  29. 29.
    Ferrara, N., and W.J. Henzel. 1989. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochemical and Biophysical Research Communications 161(2): 851–858.PubMedCrossRefGoogle Scholar
  30. 30.
    Leung, D.W., G. Cachianes, W.J. Kuang, D.V. Goeddel, and N. Ferrara. 1989. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246(4935): 1306–1309.PubMedCrossRefGoogle Scholar
  31. 31.
    Min, J., Y. Kim, E. Kim, Y. Gho, I. Kang, S. Lee, Y. Kong, and Y. Kwon. 2003. Vascular endothelial growth factor up-regulates expression of receptor activator of NF-kappa B (RANK) in endothelial cells. Concomitant increase of angiogenic responses to RANK ligand. Journal of Biological Chemistry 278(41): 39548–39557.PubMedCrossRefGoogle Scholar
  32. 32.
    Wong, B., R. Josien, S. Lee, B. Sauter, H. Li, R. Steinman, and Y. Choi. 1997. TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. The Journal of Experimental Medicine 186(12): 2075–2080.PubMedCrossRefGoogle Scholar
  33. 33.
    Leibbrandt, A., and J.M. Penninger. 2008. RANK/RANKL: regulators of immune responses and bone physiology. Annals of the New York Academy of Sciences 1143(1): 123–150.PubMedCrossRefGoogle Scholar
  34. 34.
    Mandelin, J., T.F. Li, M. Liljestrom, M. Kroon, R. Hanemaaijer, S. Santavirta, and Y.T. Konttinen. 2003. Imbalance of RANKL/RANK/OPG system in interface tissue in loosening of total hip replacement. The Journal of Bone and Joint Surgery British 85(8): 1196–1201.CrossRefGoogle Scholar
  35. 35.
    Haynes, D.R., T. Crotti, A. Potter, M. Loric, G.J. Atkins, D.W. Howie, and D.M. Findlay. 2001. The osteoclastogenic molecules RANKL and RANK are associated with periprosthetic osteolysis. The Journal of Bone and Joint Surgery. British Volume 83(6): 902–911.PubMedCrossRefGoogle Scholar
  36. 36.
    Ren, W., R. Blasier, X. Peng, T. Shi, P.H. Wooley, and D. Markel. 2009. Effect of oral erythromycin therapy in patients with aseptic loosening of joint prostheses. Bone 44(4): 671–677.PubMedCrossRefGoogle Scholar
  37. 37.
    Bylski, D., C. Wedemeyer, J. Xu, T. Sterner, F. Löer, and M. von Knoch. 2009. Alumina ceramic particles, in comparison with titanium particles, hardly affect the expression of RANK-, TNF-α-, and OPG-mRNA in the THP-1 human monocytic cell line. Journal of Biomedical Materials Research. Part A 89(3): 707–716.PubMedCrossRefGoogle Scholar
  38. 38.
    Granchi, D., G. Ciapetti, I. Amato, S. Pagani, E. Cenni, L. Savarino, S. Avnet, J. Peris, A. Pellacani, and N. Baldini. 2004. The influence of alumina and ultra-high molecular weight polyethylene particles on osteoblast–osteoclast cooperation. Biomaterials 25(18): 4037–4045.PubMedCrossRefGoogle Scholar
  39. 39.
    Baumann, B., C. Rader, J. Seufert, U. Nöth, O. Rolf, J. Eulert, and F. Jakob. 2004. Effects of polyethylene and TiAIV wear particles on expression of RANK, RANKL and OPG mRNA. Acta Orthopaedica 75(3): 295–302.CrossRefGoogle Scholar
  40. 40.
    Masui, T., S. Sakano, Y. Hasegawa, H. Warashina, and N. Ishiguro. 2005. Expression of inflammatory cytokines, RANKL and OPG induced by titanium, cobalt-chromium and polyethylene particles. Biomaterials 26(14): 1695–1702.PubMedCrossRefGoogle Scholar
  41. 41.
    Inoue, J., T. Ishida, N. Tsukamoto, N. Kobayashi, A. Naito, S. Azuma, and T. Yamamoto. 2000. Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling. Experimental Cell Research 254(1): 14.PubMedCrossRefGoogle Scholar
  42. 42.
    Rauner, M., W. Sipos, and P. Pietschmann. 2006. Osteoimmunology. International Archives of Allergy and Immunology 143(1): 31–48.PubMedCrossRefGoogle Scholar
  43. 43.
    Hongming, H., and H. Jian. 2009. Bortezomib inhibits maturation and function of osteoclasts from PBMCs of patients with multiple myeloma by downregulating TRAF6. Leukemia Research 33(1): 115–122.PubMedCrossRefGoogle Scholar
  44. 44.
    Ang, E., N.J. Pavlos, S.L. Rea, M. Qi, T. Chai, J.P. Walsh, T. Ratajczak, M.H. Zheng, and J. Xu. 2009. Proteasome inhibitors impair RANKL-induced NF-κB activity in osteoclast-like cells via disruption of p62, TRAF6, CYLD, and IκBα signaling cascades. Journal of Cellular Physiology 220(2): 450–459.PubMedCrossRefGoogle Scholar
  45. 45.
    Baldwin, L., B. Flanagan, P. McLaughlin, R. Parkinson, J. Hunt, and D. Williams. 2002. A study of tissue interface membranes from revision accord knee arthroplasty: the role of T lymphocytes. Biomaterials 23(14): 3007–3014.PubMedCrossRefGoogle Scholar
  46. 46.
    Micklem, K., E. Rigney, J. Cordell, D. Simmons, P. Stross, H. Turley, B. Seed, and D. Mason. 1989. A human macrophage-associated antigen (CD68) detected by six different monoclonal antibodies. British Journal of Haematology 73(1): 6–11.PubMedCrossRefGoogle Scholar
  47. 47.
    Anan, A., E.S. Baskin-Bey, H. Isomoto, J.L. Mott, S.F. Bronk, J.H. Albrecht, and G.J. Gores. 2006. Proteasome inhibition attenuates hepatic injury in the bile duct-ligated mouse. American Journal of Physiology - Gastrointestinal and Liver Physiology 291(4): G709–G716.PubMedCrossRefGoogle Scholar
  48. 48.
    Pagliari, L.J., H. Perlman, H. Liu, and R.M. Pope. 2000. Macrophages require constitutive NF-kappa B activation to maintain A1 expression and mitochondrial homeostasis. Molecular and Cellular Biology 20(23): 8855–8865.PubMedCrossRefGoogle Scholar
  49. 49.
    Landgraeber, S., M. von Knoch, F. Loer, A. Wegner, M. Tsokos, and M. Totsch. 2008. Extrinsic and intrinsic pathways of apoptosis in aseptic loosening after total hip replacement. Biomaterials 29(24–25): 3444–3450.PubMedCrossRefGoogle Scholar
  50. 50.
    Mao, X., Pan, X., Cheng, T., and Zhang, X. 2011. Therapeutic potential of the proteasome inhibitor bortezomib on titanium particle-induced inflammation in a murine model. Inflammation. Available from URL: (doi:10.1007/s10753-011-9392-7).
  51. 51.
    Jimi, E., K. Aoki, H. Saito, F. D'Acquisto, M.J. May, I. Nakamura, T. Sudo, T. Kojima, F. Okamoto, and H. Fukushima. 2004. Selective inhibition of NF-kappaB blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo. Nature Medicine 10(6): 617–624.PubMedCrossRefGoogle Scholar
  52. 52.
    Hofbauer, L., D. Lacey, C. Dunstan, T. Spelsberg, B. Riggs, and S. Khosla. 1999. Interleukin-1 [beta] and tumor necrosis factor-[alpha], but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 25(3): 255–259.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Xin Mao
    • 1
  • Xiaoyun Pan
    • 1
  • Song Zhao
    • 1
  • Xiaochun Peng
    • 1
  • Tao Cheng
    • 1
  • Xianlong Zhang
    • 1
  1. 1.Department of OrthopaedicsThe Sixth Affiliated People’s Hospital, Shanghai Jiaotong University School of MedicineShanghaiChina

Personalised recommendations