Skip to main content

Macrophages’ contribution to ectopic osteogenesis in combination with blood clot and bone substitute: possibility for application in bone regeneration strategies

International Orthopaedics volume 45, pages 1087–1095 (2021)Cite this article

Abstract

Purpose

Given the great potential of macrophages in the processes of tissue repair and regeneration, the aim of our study was to examine the contribution that macrophages will have in osteogenic process when combined and implanted with blood clot (BC) and mineral bone substitute (MBS) in mice subcutaneous implantation model.

Methods

Three types of implants were constructed and implanted subcutaneously into BALB/c mice: (1) RMBM implants (made of resident tissue macrophages, BC and MBS), (2) BM implants (made of BC and MBS), and (3) M implants (made of MBS only) where the last two served as control implants. One, two, four and eight weeks after implantation implants were explanted, and histochemical, immunohistochemical, and histomorphometric analyses were performed.

Results

Increased vascularization, particularly pronounced two and four weeks after implantation and pronounced tissue infiltration in eight week term in RMBM implants compared with both other types, likewise the presence of osteoblast-like cells, osteoid-like structures, and more prominent osteopontin and osteocalcin immunoexpression in RMBM implants indicated more pronounced osteogenic process within them.

Conclusion

Our results suggest that macrophages deserve to be considered as a cell component when constructing implants in bone regenerative medicine strategies to improve bone fracture healing process.

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

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

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

Data availability

The datasets used in the current study are available from the corresponding author on reasonable request.

References

  1. Crowther M, Brown NJ, Bishop ET, Lewis CE (2001) Microenvironmental influence on macrophage regulation of angiogenesis in wounds and malignant tumors. J Leukoc Biol 70(4):478–490. https://doi.org/10.1189/jlb.70.4.478

    Article  CAS  PubMed  Google Scholar 

  2. Marsell R, Einhorn TA (2011) The biology of fracture healing. Injury 42(6):551–555. https://doi.org/10.1016/j.injury.2011.03.031

    Article  PubMed  PubMed Central  Google Scholar 

  3. Najdanović JG, Cvetković VJ, Stojanović S, Vukelić-Nikolić MĐ, Stanisavljević MN, Živković JM, Najman SJ (2015) The influence of adipose-derived stem cells induced into endothelial cells on ectopic vasculogenesis and osteogenesis. Cell Mol Bioeng 8:577–590. https://doi.org/10.1007/s12195-015-0403-x

    Article  CAS  Google Scholar 

  4. Sinder BP, Pettit AR, McCauley LK (2015) Macrophages: their emerging roles in bone. J Bone Miner Res 30(12):2140–2149. https://doi.org/10.1002/jbmr.2735

    Article  PubMed  Google Scholar 

  5. Xia Z, Triffitt JT (2006) A review on macrophage responses to biomaterials. Biomed Mater 1(1):R1–R9. https://doi.org/10.1088/1748-6041/1/1/R01

    Article  CAS  PubMed  Google Scholar 

  6. Schmidt-Bleek K, Schell H, Schulz N, Hoff P, Perka C, Buttgereit F, Volk HD, Lienau J, Duda GN (2012) Inflammatory phase of bone healing initiates the regenerative healing cascade. Cell Tissue Res 347(3):567–573. https://doi.org/10.1007/s00441-011-1205-7

    Article  CAS  PubMed  Google Scholar 

  7. Dong L, Wang C (2013) Harnessing the power of macrophages/monocytes for enhanced bone tissue engineering. Trends Biotechnol 31(6):342–346. https://doi.org/10.1016/j.tibtech.2013.04.001

    Article  CAS  PubMed  Google Scholar 

  8. Haines NM, Lack WD, Seymour RB, Bosse MJ (2016) Defining the lower limit of a “critical bone defect” in open diaphyseal tibial fractures. J Orthop Trauma 30(5):e158–e163

    Article  PubMed  Google Scholar 

  9. Li D, Li M, Liu P, Zhang Y, Lu J, Li J (2014) Tissue-engineered bone constructed in a bioreactor for repairing critical-sized bone defects in sheep. Int Orthop 38(11):2399–2406. https://doi.org/10.1007/s00264-014-2389-8

    Article  PubMed  Google Scholar 

  10. Vukicevic S, Oppermann H, Verbanac D, Jankolija M, Popek I, Curak J, Brkljacic J, Pauk M, Erjavec I, Francetic I, Dumic-Cule I, Jelic M, Durdevic D, Vlahovic T, Novak R, Kufner V, Bordukalo Niksic T, Kozlovic M, Banic Tomisic Z, Bubic-Spoljar J, Bastalic I, Vikic-Topic S, Peric M, Pecina M, Grgurevic L (2014) The clinical use of bone morphogenetic proteins revisited: a novel biocompatible carrier device OSTEOGROW for bone healing. Int Orthop 38(3):635–647. https://doi.org/10.1007/s00264-013-2201-1

    Article  PubMed  Google Scholar 

  11. Dumic-Cule I, Peric M, Kucko L, Grgurevic L, Pecina M, Vukicevic S (2018) Bone morphogenetic proteins in fracture repair. Int Orthop 42(11):2619–2626. https://doi.org/10.1007/s00264-018-4153-y

    Article  PubMed  Google Scholar 

  12. Zhang H, Yang L, Yang XG, Wang F, Feng JT, Hua KC, Li Q, Hu YC (2019) Demineralized bone matrix carriers and their clinical applications: an overview. Orthop Surg 11(5):725–737. https://doi.org/10.1111/os.12509

    Article  PubMed  PubMed Central  Google Scholar 

  13. Hoemann CD, Hurtig M, Rossomacha E, Sun J, Chevrier A, Shive MS, Buschmann MD (2005) Chitosan-glycerol phosphate/blood implants improve hyaline cartilage repair in ovine microfracture defects. J Bone Joint Surg Am 87(12):2671–2686

    Article  PubMed  Google Scholar 

  14. Cvetković VJ, Najdanović JG, Vukelić-Nikolić MĐ, Stojanović S, Najman SJ (2015) Osteogenic potential of in vitro osteo-induced adipose-derived mesenchymal stem cells combined with platelet-rich plasma in an ectopic model. Int Orthop 39(11):2173–2180. https://doi.org/10.1007/s00264-015-2929-x

    Article  PubMed  Google Scholar 

  15. Živković JM, Najman SJ, Vukelić MĐ, Stojanović S, Aleksić MV, Stanisavljević MN, Najdanović JG (2015) Osteogenic effect of inflammatory macrophages loaded onto mineral bone substitute in subcutaneous implants. Arch Biol Sci 67(1):173–186. https://doi.org/10.2298/ABS140915020Z

    Article  Google Scholar 

  16. Najman SJ, Cvetković VJ, Najdanović JG, Stojanović S, Vukelić-Nikolić MĐ, Vučković I, Petrović D (2016) Ectopic osteogenic capacity of freshly isolated adipose-derived stromal vascular fraction cells supported with platelet-rich plasma: a simulation of intraoperative procedure. J Craniomaxillofac Surg 44(10):1750–1760. https://doi.org/10.1016/j.jcms.2016.08.011

    Article  PubMed  Google Scholar 

  17. Noori A, Ashrafi SJ, Vaez-Ghaemi R, Hatamian-Zaremi A, Webster TJ (2017) A review of fibrin and fibrin composites for bone tissue engineering. Int J Nanomedicine 12:4937–4961. https://doi.org/10.2147/IJN.S124671

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vukelić-Nikolić MĐ, Najman SJ, Vasiljević PJ, Jevtović-Stoimenov TM, Cvetković VJ, Andrejev MN, Mitić ŽJ (2018) Osteogenic capacity of diluted platelet-rich plasma in ectopic bone-forming model: benefits for bone regeneration. J Craniomaxillofac Surg 46(11):1911–1918. https://doi.org/10.1016/j.jcms.2018.09.005

    Article  PubMed  Google Scholar 

  19. Dumic-Cule I, Pecina M, Jelic M, Jankolija M, Popek I, Grgurevic L, Vukicevic S (2015) Biological aspects of segmental bone defects management. Int Orthop 39(5):1005–1011. https://doi.org/10.1007/s00264-015-2728-4

    Article  PubMed  Google Scholar 

  20. Minutti CM, Knipper JA, Allen JE, Zaiss DM (2017) Tissue-specific contribution of macrophages to wound healing. Semin Cell Dev Biol 61:3–11. https://doi.org/10.1016/j.semcdb.2016.08.006

    Article  CAS  PubMed  Google Scholar 

  21. Hao W, Dong J, Jiang M, Wu J, Cui F, Zhou D (2010) Enhanced bone formation in large segmental radial defects by combining adipose-derived stem cells expressing bone morphogenetic protein 2 with nHA/RHLC/PLA scaffold. Int Orthop 34(8):1341–1349. https://doi.org/10.1007/s00264-009-0946-3

    Article  PubMed  PubMed Central  Google Scholar 

  22. Pang H, Wu XH, Fu SL, Luo F, Zhang ZH, Hou TY, Li ZQ, Chang ZQ, Yu B, Xu JZ (2013) Prevascularisation with endothelial progenitor cells improved restoration of the architectural and functional properties of newly formed bone for bone reconstruction. Int Orthop 37(4):753–759. https://doi.org/10.1007/s00264-012-1751-y

    Article  PubMed  PubMed Central  Google Scholar 

  23. Barbeck M, Najman S, Stojanović S, Mitić Ž, Živković JM, Choukroun J, Kovačević P, Sader R, Kirkpatrick CJ, Ghanaati S (2015) Addition of blood to a phycogenic bone substitute leads to increased in vivo vascularization. Biomed Mater 10(5):055007. https://doi.org/10.1088/1748-6041/10/5/055007

    Article  CAS  PubMed  Google Scholar 

  24. Scull CM, Hays WD, Fischer TH (2010) Macrophage pro-inflammatory cytokine secretion is enhanced following interaction with autologous platelets. J Inflamm (Lond) 7:53. https://doi.org/10.1186/1476-9255-7-53

    Article  CAS  Google Scholar 

  25. Kahles F, Findeisen HM, Bruemmer D (2014) Osteopontin: a novel regulator at the cross roads of inflammation, obesity and diabetes. Mol Metab 3(4):384–393. https://doi.org/10.1016/j.molmet.2014.03.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yu B, Zhang Y, Li X, Wang Q, Ouyang Y, Xia Y, Yu B, Lin B, Li S, Fan Y, Chen Y (2013) The use of injectable chitosan/nanohydroxyapatite/collagen composites with bone marrow mesenchymal stem cells to promote ectopic bone formation in vivo. J Nanomater 2013:506593. https://doi.org/10.1155/2013/506593

    Article  CAS  Google Scholar 

  27. Carvalho MS, Cabral JM, da Silva CL, Vashishth D (2019) Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation, osteogenic differentiation, and angiogenic properties. J Cell Biochem 120(4):6555–6569. https://doi.org/10.1002/jcb.27948

    Article  CAS  PubMed  Google Scholar 

  28. Luo ML, Jiao Y, Gong WP, Li Y, Niu LN, Tay FR, Chen JH (2020) Macrophages enhance mesenchymal stem cell osteogenesis via down-regulation of reactive oxygen species. J Dent 94:103297. https://doi.org/10.1016/j.jdent.2020.103297

    Article  CAS  PubMed  Google Scholar 

  29. Bhat A, Wooten RM, Jayasuriya AC (2013) Secretion of growth factors from macrophages when cultured with microparticles. J Biomed Mater Res A 101(11):3170–3180. https://doi.org/10.1002/jbm.a.34604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sartori M, Giavaresi G, Parrilli A, Ferrari A, Aldini NN, Morra M, Cassinelli C, Bollati D, Fini M (2015) Collagen type I coating stimulates bone regeneration and osteointegration of titanium implants in the osteopenic rat. Int Orthop 39(10):2041–2052. https://doi.org/10.1007/s00264-015-2926-0

    Article  PubMed  Google Scholar 

  31. Rutkovskiy A, Stensløkken KO, Vaage IJ (2016) Osteoblast differentiation at a glance. Med Sci Monit Basic Res 22:95–106

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This study was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Project Grant No. III41017) and Faculty of Medicine, University of Niš, Serbia (Internal Project No. 16).

Author information

Authors and Affiliations

  1. Department of Biology and Human Genetics, Faculty of Medicine, University of Niš, Niš, 18108, Serbia

    Jelena M. Živković, Sanja T. Stojanović, Marija Đ. Vukelić-Nikolić, Jelena G. Najdanović & Stevo J. Najman

  2. Department for Cell and Tissue Engineering, Scientific Research Center for Biomedicine, Faculty of Medicine, University of Niš, Niš, 18108, Serbia

    Jelena M. Živković, Sanja T. Stojanović, Marija Đ. Vukelić-Nikolić, Milena B. Radenković, Jelena G. Najdanović & Stevo J. Najman

  3. Institute of Physiology, Faculty of Medicine, University of Niš, Niš, 18108, Serbia

    Milan Ćirić

Authors
  1. Jelena M. Živković
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sanja T. Stojanović
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marija Đ. Vukelić-Nikolić
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Milena B. Radenković
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jelena G. Najdanović
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Milan Ćirić
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Stevo J. Najman
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jelena M. Živković.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in study involving animals were in accordance with the ethical standards and have been approved by the Faculty of Medicine University of Niš (approval number 01-5518-6).

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Živković, J.M., Stojanović, S.T., Vukelić-Nikolić, M.Đ. et al. Macrophages’ contribution to ectopic osteogenesis in combination with blood clot and bone substitute: possibility for application in bone regeneration strategies. International Orthopaedics (SICOT) 45, 1087–1095 (2021). https://doi.org/10.1007/s00264-020-04826-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00264-020-04826-0

Keywords

  • Macrophages
  • Blood clot
  • Ectopic osteogenesis
  • Subcutaneous implantation
  • Implant
  • Mineral bone substitute