Skip to main content

Subtypes of endothelial progenitor cells affect healing of segmental bone defects differently

We’re sorry, something doesn't seem to be working properly.

Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.



Treating fracture nonunion with endothelial progenitor cells (EPCs) is a promising approach. Nevertheless, the effect of different EPC-related cell populations remains unclear. In this study, we compared the therapeutic potential of early (E-EPCs) and late EPCs (L-EPCs).


Male Fischer 344 rats were used for cell isolation and in vivo experiments. Bone marrow-derived E-EPCs and L-EPCs were kept in culture for seven to ten days and four weeks, respectively. For each treatment group, we seeded one million cells on a gelatin scaffold before implantation in a segmental defect created in a rat femur; control animals received a cell-free scaffold. Bone healing was monitored via radiographs for up to ten weeks after surgery. In vitro, secretion of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2 was quantified by ELISA for both cell populations. Tube formation assays were also performed.


Final radiographs showed complete (four out of five rats) or partial (one out of five rats) union with E-EPC treatment. In contrast, complete healing was achieved in only one of five animals after L-EPC implantation, while control treatment resulted in nonunion in all animals. In vitro, E-EPCs released more VEGF, but less BMP-2 than L-EPCs. In addition, L-EPCs formed longer and more mature tubules on basement membrane matrix than E-EPCs. However, co-culture with primary osteoblasts stimulated tubulogenesis of E-EPCs while inhibiting that of L-EPCs.


We demonstrated that bone marrow-derived E-EPCs are a better alternative than L-EPCs for treatment of nonunion. We hypothesize that the expression profile of E-EPCs and their adaptation to the local environment contribute to superior bone healing.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Antonova E, Le TK, Burge R, Mershon J (2013) Tibia shaft fractures: costly burden of nonunions. BMC Musculoskelet Disord 14:42.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Sen MK, Miclau T (2007) Autologous iliac crest bone graft: should it still be the gold standard for treating nonunions? Injury 38(Suppl 1):S75–S80.

    Article  PubMed  Google Scholar 

  3. 3.

    Kim DH, Rhim R, Li L, Martha J, Swaim BH, Banco RJ, Jenis LG, Tromanhauser SG (2009) Prospective study of iliac crest bone graft harvest site pain and morbidity. Spine J 9(11):886–892.

    Article  PubMed  Google Scholar 

  4. 4.

    Hankenson KD, Dishowitz M, Gray C, Schenker M (2011) Angiogenesis in bone regeneration. Injury 42(6):556–561.

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Asahara T, Kawamoto A, Masuda H (2011) Concise review: circulating endothelial progenitor cells for vascular medicine. Stem Cells 29(11):1650–1655.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Atesok K, Li R, Stewart DJ, Schemitsch EH (2010) Endothelial progenitor cells promote fracture healing in a segmental bone defect model. J Orthop Res 28(8):1007–1014.

    PubMed  Google Scholar 

  7. 7.

    Medina RJ, O’Neill CL, Sweeney M, Guduric-Fuchs J, Gardiner TA, Simpson DA, Stitt AW (2010) Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities. BMC Med Genet 3:18.

    Google Scholar 

  8. 8.

    Declercq HA, De Ridder LI, Cornelissen MJ (2005) Isolation and Osteogenic differentiation of rat Periosteum-derived cells. Cytotechnology 49(1):39–50.

    Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Eman RM, Meijer HA, Oner FC, Dhert WJ, Alblas J (2016) Establishment of an early vascular network promotes the formation of ectopic bone. Tissue Eng A 22(3–4):253–262.

    CAS  Article  Google Scholar 

  10. 10.

    Ikutomi M, Sahara M, Nakajima T, Minami Y, Morita T, Hirata Y, Komuro I, Nakamura F, Sata M (2015) Diverse contribution of bone marrow-derived late-outgrowth endothelial progenitor cells to vascular repair under pulmonary arterial hypertension and arterial neointimal formation. J Mol Cell Cardiol 86:121–135.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Yang YQ, Tan YY, Wong R, Wenden A, Zhang LK, Rabie AB (2012) The role of vascular endothelial growth factor in ossification. Int J Oral Sci 4(2):64–68.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Rosen V (2009) BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev 20(5–6):475–480.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Bai Y, Li P, Yin G, Huang Z, Liao X, Chen X, Yao Y (2013) BMP-2, VEGF and bFGF synergistically promote the osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. Biotechnol Lett 35(3):301–308.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Li P, Bai Y, Yin G, Pu X, Huang Z, Liao X, Chen X, Yao Y (2014) Synergistic and sequential effects of BMP-2, bFGF and VEGF on osteogenic differentiation of rat osteoblasts. J Bone Miner Metab 32(6):627–635.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Garrison KR, Shemilt I, Donell S, Ryder JJ, Mugford M, Harvey I, Song F, Alt V (2010) Bone morphogenetic protein (BMP) for fracture healing in adults. Cochrane Database Syst Rev 6:CD006950.

    Google Scholar 

  16. 16.

    Street J, Bao M, deGuzman L, Bunting S, Peale FV Jr, Ferrara N, Steinmetz H, Hoeffel J, Cleland JL, Daugherty A, van Bruggen N, Redmond HP, Carano RA, Filvaroff EH (2002) Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A 99(15):9656–9661.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Li R, Stewart DJ, von Schroeder HP, Mackinnon ES, Schemitsch EH (2009) Effect of cell-based VEGF gene therapy on healing of a segmental bone defect. J Orthop Res 27(1):8–14.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Bouletreau PJ, Warren SM, Spector JA, Peled ZM, Gerrets RP, Greenwald JA, Longaker MT (2002) Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: implications for fracture healing. Plast Reconstr Surg 109(7):2384–2397

    Article  PubMed  Google Scholar 

  19. 19.

    Li R, Nauth A, Gandhi R, Syed K, Schemitsch EH (2014) BMP-2 mRNA expression after endothelial progenitor cell therapy for fracture healing. J Orthop Trauma 28(Suppl 1):S24–S27.

    Article  PubMed  Google Scholar 

  20. 20.

    Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM (1999) VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 18(14):3964–3972.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Einhorn TA, Gerstenfeld LC (2015) Fracture healing: mechanisms and interventions. Nat Rev Rheumatol 11(1):45–54.

    Article  PubMed  Google Scholar 

  22. 22.

    Mukai N, Akahori T, Komaki M, Li Q, Kanayasu-Toyoda T, Ishii-Watabe A, Kobayashi A, Yamaguchi T, Abe M, Amagasa T, Morita I (2008) A comparison of the tube forming potentials of early and late endothelial progenitor cells. Exp Cell Res 314(3):430–440.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Spigoni V, Picconi A, Cito M, Ridolfi V, Bonomini S, Casali C, Zavaroni I, Gnudi L, Metra M, Dei Cas A (2012) Pioglitazone improves in vitro viability and function of endothelial progenitor cells from individuals with impaired glucose tolerance. PLoS One 7(11):e48283.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Fuchs S, Ghanaati S, Orth C, Barbeck M, Kolbe M, Hofmann A, Eblenkamp M, Gomes M, Reis RL, Kirkpatrick CJ (2009) Contribution of outgrowth endothelial cells from human peripheral blood on in vivo vascularization of bone tissue engineered constructs based on starch polycaprolactone scaffolds. Biomaterials 30(4):526–534.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Tura O, Skinner EM, Barclay GR, Samuel K, Gallagher RC, Brittan M, Hadoke PW, Newby DE, Turner ML, Mills NL (2013) Late outgrowth endothelial cells resemble mature endothelial cells and are not derived from bone marrow. Stem Cells 31(2):338–348.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Minami Y, Nakajima T, Ikutomi M, Morita T, Komuro I, Sata M, Sahara M (2015) Angiogenic potential of early and late outgrowth endothelial progenitor cells is dependent on the time of emergence. Int J Cardiol 186:305–314.

    Article  PubMed  Google Scholar 

  27. 27.

    Guan XM, Cheng M, Li H, Cui XD, Li X, Wang YL, Sun JL, Zhang XY (2013) Biological properties of bone marrow-derived early and late endothelial progenitor cells in different culture media. Mol Med Rep 8(6):1722–1728.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Sekiguchi H, Ii M, Losordo DW (2009) The relative potency and safety of endothelial progenitor cells and unselected mononuclear cells for recovery from myocardial infarction and ischemia. J Cell Physiol 219(2):235–242.

    CAS  Article  PubMed  Google Scholar 

Download references


We would like to thank Sarah Desjardins for the critical reading of the manuscript.


This study was funded by the Orthopedic Trauma Association (OTA) and the Canadian Institutes of Health Research (CIHR; MOP-115111).

Author information



Corresponding author

Correspondence to Aaron Nauth.

Ethics declarations

Ethical approval

All procedures performed in this study involving animals were in accordance with the ethical standards of the institution at which the study was conducted.

Conflict of interest

Erica M. Giles, Charles Godbout, Wendy Chi, Michael A. Glick, Tony Lin, Ru Li, Emil H. Schemitsch, and Aaron Nauth declare that they have no conflict of interest.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Giles, E.M., Godbout, C., Chi, W. et al. Subtypes of endothelial progenitor cells affect healing of segmental bone defects differently. International Orthopaedics (SICOT) 41, 2337–2343 (2017).

Download citation


  • Bone
  • Stem cell therapy
  • Endothelial progenitor cells
  • Fracture healing
  • Nonunion
  • Tissue engineering