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Human immune cells' behavior and survival under bioenergetically restricted conditions in an in vitro fracture hematoma model

Abstract

The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma.

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References

  1. Mercier FE, Ragu C, Scadden DT . The bone marrow at the crossroads of blood and immunity. Nat Rev Immunol 2012; 12: 49–60.

    Article  CAS  Google Scholar 

  2. Teti A . Bone development: overview of bone cells and signaling. Curr Osteoporos Rep 2011; 9: 264–273.

    Article  Google Scholar 

  3. McKibbin B . The biology of fracture healing in long bones. J Bone Joint Surg Br 1978; 60-B: 150–162.

    Article  CAS  Google Scholar 

  4. Remedios A . Bone and bone healing. Vet Clin North Am Small Anim Pract 1999; 29: 1029–1044.

    Article  CAS  Google Scholar 

  5. Naik AA, Xie C, Zuscik MJ, Kingsley P, Schwarz EM, Awad H et al. Reduced COX-2 expression in aged mice is associated with impaired fracture healing. J Bone Miner Res 2009; 24: 251–264.

    Article  CAS  Google Scholar 

  6. Gerstenfeld LC, Thiede M, Seibert K, Mielke C, Phippard D, Svagr B et al. Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs. J Orthop Res 2003; 21: 670–675.

    Article  CAS  Google Scholar 

  7. Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O'Keefe RJ . Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 2002; 109: 1405–1415.

    Article  CAS  Google Scholar 

  8. Kolar P, Schmidt-Bleek K, Schell H, Gaber T, Toben D, Schmidmaier G et al. The early fracture hematoma and its potential role in fracture healing. Tissue Eng Part B Rev 2010; 16: 427–434.

    Article  Google Scholar 

  9. Schaffer M, Barbul A . Lymphocyte function in wound healing and following injury. Br J Surg 1998; 85: 444–460.

    Article  CAS  Google Scholar 

  10. Simpson DM, Ross R . The neutrophilic leukocyte in wound repair a study with antineutrophil serum. J Clin Invest 1972; 51: 2009–2023.

    Article  CAS  Google Scholar 

  11. Park JE, Barbul A . Understanding the role of immune regulation in wound healing. Am J Surg 2004; 187: 11S–16S.

    Article  CAS  Google Scholar 

  12. Efron JE, Frankel HL, Lazarou SA, Wasserkrug HL, Barbul A . Wound healing and T-lymphocytes. J Surg Res 1990; 48: 460–463.

    Article  CAS  Google Scholar 

  13. Toben D, Schroeder I, El Khassawna T, Mehta M, Hoffmann JE, Frisch JT et al. Fracture healing is accelerated in the absence of the adaptive immune system. J Bone Miner Res 2011; 26: 113–124.

    Article  CAS  Google Scholar 

  14. Grundnes O, Reikeras O . The importance of the hematoma for fracture healing in rats. Acta Orthop Scand 1993; 64: 340–342.

    Article  CAS  Google Scholar 

  15. Andrew JG, Andrew SM, Freemont AJ, Marsh DR . Inflammatory cells in normal human fracture healing. Acta Orthop Scand 1994; 65: 462–466.

    Article  CAS  Google Scholar 

  16. Chung R, Cool JC, Scherer MA, Foster BK, Xian CJ . Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats. J Leukoc Biol 2006; 80: 1272–1280.

    Article  CAS  Google Scholar 

  17. Schmidt-Bleek K, Schell H, Kolar P, Pfaff M, Perka C, Buttgereit F et al. Cellular composition of the initial fracture hematoma compared to a muscle hematoma: a study in sheep. J Orthop Res 2009; 27: 1147–1151.

    Article  Google Scholar 

  18. Dziurla R, Gaber T, Fangradt M, Hahne M, Tripmacher R, Kolar P et al. Effects of hypoxia and/or lack of glucose on cellular energy metabolism and cytokine production in stimulated human CD4+ T lymphocytes. Immunol Lett 2010; 131: 97–105.

    Article  CAS  Google Scholar 

  19. Tripmacher R, Gaber T, Dziurla R, Haupl T, Erekul K, Grutzkau A et al. Human CD4+ T cells maintain specific functions even under conditions of extremely restricted ATP production. Eur J Immunol 2008; 38: 1631–1642.

    Article  CAS  Google Scholar 

  20. Cramer T, Yamanishi Y, Clausen BE, Forster I, Pawlinski R, Mackman N et al. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell 2003; 112: 645–657.

    Article  CAS  Google Scholar 

  21. Kolar P, Gaber T, Perka C, Duda GN, Buttgereit F . Human Early Fracture hematoma is characterized by inflammation and hypoxia. Clin Orthop Relat Res 2011; 469: 3118–3126.

    Article  Google Scholar 

  22. Hoff P, Gaber T, Schmidt-Bleek K, Senturk U, Tran CL, Blankenstein K et al. Immunologically restricted patients exhibit a pronounced inflammation and inadequate response to hypoxia in fracture hematomas. Immunol Res 2011; 51: 116–122.

    Article  CAS  Google Scholar 

  23. Cornell CN, Lane JM . Newest factors in fracture healing. Clin Orthop Relat Res 1992; 277: 297–311.

    Google Scholar 

  24. Hauser CJ, Zhou X, Joshi P, Cuchens MA, Kregor P, Devidas M et al. The immune microenvironment of human fracture/soft-tissue hematomas and its relationship to systemic immunity. J Trauma 1997; 42: 895–903.

    Article  CAS  Google Scholar 

  25. Gaber T, Dziurla R, Tripmacher R, Burmester GR, Buttgereit F . Hypoxia inducible factor (HIF) in rheumatology: low O2! See what HIF can do! Ann Rheum Dis 2005; 64: 971–980.

    Article  CAS  Google Scholar 

  26. Schipani E, Maes C, Carmeliet G, Semenza GL . Regulation of osteogenesis-angiogenesis coupling by HIFs and VEGF. J Bone Miner Res 2009; 24: 1347–1353.

    Article  CAS  Google Scholar 

  27. Behr B, Tang C, Germann G, Longaker MT, Quarto N . Locally applied vascular endothelial growth factor a increases the osteogenic healing capacity of human adipose-derived stem cells by promoting osteogenic and endothelial differentiation. Stem Cells 2011; 29: 286–296.

    Article  CAS  Google Scholar 

  28. Martin D, Galisteo R, Gutkind JS . CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex. J Biol Chem 2009; 284: 6038–6042.

    Article  CAS  Google Scholar 

  29. Rosenkilde MM, Schwartz TW . The chemokine system—a major regulator of angiogenesis in health and disease. APMIS 2004; 112: 481–495.

    Article  CAS  Google Scholar 

  30. Bastian O, Pillay J, Alblas J, Leenen L, Koenderman L, Blokhuis T . Systemic inflammation and fracture healing. J Leukoc Biol 2011; 89: 669–673.

    Article  CAS  Google Scholar 

  31. Yadav A, Saini V, Arora S . MCP-1: chemoattractant with a role beyond immunity: a review. Clin Chim Acta 2010; 411: 1570–1579.

    Article  CAS  Google Scholar 

  32. Street J, Winter D, Wang JH, Wakai A, McGuinness A, Redmond HP . Is human fracture hematoma inherently angiogenic? Clin Orthop Relat Res 2000; 378: 224–237.

    Article  Google Scholar 

  33. Arnold R, Brenner D, Becker M, Frey CR, Krammer PH . How T lymphocytes switch between life and death. Eur J Immunol 2006; 36: 1654–1658.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Manuela Jakstadt for her excellent technical assistance. This work was supported in part by the BCRT-Grant I.

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Correspondence to Paula Hoff.

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Hoff, P., Maschmeyer, P., Gaber, T. et al. Human immune cells' behavior and survival under bioenergetically restricted conditions in an in vitro fracture hematoma model. Cell Mol Immunol 10, 151–158 (2013). https://doi.org/10.1038/cmi.2012.56

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  • DOI: https://doi.org/10.1038/cmi.2012.56

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