Skip to main content

Wnt3a and ASCs are capable of restoring mineralization in staph aureus-infected primary murine osteoblasts

Abstract

Introduction

Bone infections are one of the main reasons for impaired bone regeneration and non-union formation. In previous experimental animal studies we could already demonstrate that bone defects due to prior infections showed a markedly reduced healing capacity, which could effectively be enhanced via application of Wnt3a and Adipose-derived stromal cells (ASCs). For a more in-depth analysis, we investigated proliferation and mineralization of cultured osteoblasts infected with staph aureus and sought to investigate effects of Wnt3a and ASCs on infected osteoblasts.

Materials and methods

Primary murine osteoblasts were isolated from calvariae and infected with staph aureus. Infected osteoblasts received treatment via application of recombinant Wnt3a, ASC conditioned medium and were furthermore cocultured with ASCs. Osteoblasts were evaluated by Alamar blue assay for metabolic activity, TUNEL-assay for apoptosis, ALP and Alizarin Red staining for mineralization. In addition, immunoflourescent staining (IF) and qRT-PCR analyses were performed.

Results

Infected osteoblasts showed a markedly reduced ability for mineralization and increased apoptosis, which could be restored to physiological levels by Wnt3a and ASC treatment. Interestingly, metabolic activity of osteoblasts seemed to be unaffected by staph aureus infection. Additional analyses of Wnt-pathway activity revealed effective enhancement of canonical Wnt-pathway activity in Wnt3a-treated osteoblasts.

Conclusions

In summary, we gained further osteoblast-related insights into pathomechanisms of reduced bone healing capacity upon infections.

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

Fig. 1
Fig. 2
Fig. 3

Abbreviations

ASC:

Adipose-derived stromal cell

RANKL:

Receptor activator nuclear factor-κB ligand

OPG:

Osteoprotegerin

ALP:

Alkaline phospatase

GSK-3β :

Glycogen synthase kinase 3 beta

sFRP1:

Secreted frizzled-related protein 1

TRAIL:

Tumor necrosis factor-related apoptosis-inducing ligand

BMSC:

Bone marrow-derived stem cells

Runx 2:

Runt-related transcription factor 2

References

  1. 1.

    Zimmermann G, Wagner C, Schmeckenbrecher K, Wentzensen A, Moghaddam A (2009) Treatment of tibial shaft non-unions: bone morphogenetic proteins versus autologous bone graft. Injury 40:50–53

    Article  Google Scholar 

  2. 2.

    Lew DP, Waldvogel FA (2004) Osteomyelitis. Lancet 364:369–379. https://doi.org/10.1016/s0140-6736(04)16727-5

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Hudson MC, Ramp WK, Nicholson NC, Williams AS, Nousiainen MT (1995) Internalization of Staphylococcus aureus by cultured osteoblasts (in eng). Microb Pathog 19:409–419. https://doi.org/10.1006/mpat.1995.0075

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Valour F, Rasigade JP, Trouillet-Assant S, Gagnaire J, Bouaziz A, Karsenty J, Lacour C, Bes M, Lustig S, Bénet T, Chidiac C, Etienne J, Vandenesch F, Ferry T, Laurent F, Lyon BJISG (2015) Delta-toxin production deficiency in Staphylococcus aureus: a diagnostic marker of bone and joint infection chronicity linked with osteoblast invasion and biofilm formation (in eng). Clin Microbiol Infect 21:568.e1. https://doi.org/10.1016/j.cmi.2015.01.026

    CAS  Article  Google Scholar 

  5. 5.

    Bar-Shavit Z (2008) Taking a toll on the bones: regulation of bone metabolism by innate immune regulators (in eng). Autoimmunity 41:195–203. https://doi.org/10.1080/08916930701694469

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Claro T, Widaa A, Mc Donell C, Foster TJ, O’brien FJ, Kerrigan SW, (2013) Staphylococcus aureus protein A binding to osteoblast tumour necrosis factor receptor 1 results in activation of nuclear factor kappa B and release of interleukin-6 in bone infection. Microbiology 159:147–154. https://doi.org/10.1099/mic.0.063016-0

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Josse J, Guillaume C, Bour C, Lemaire F, Mongaret C, Draux F, Velard F, Gangloff SC (2016) Impact of the maturation of human primary bone-forming cells on their behavior in acute or persistent staphylococcus aureus infection models (in eng). Front Cell Infect Microbiol 6:64–64. https://doi.org/10.3389/fcimb.2016.00064

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Bost KL, Ramp WK, Nicholson NC, Bento JL, Marriott I, Hudson MC (1999) Staphylococcus aureus infection of mouse or human osteoblasts induces high levels of interleukin-6 and interleukin-12 production (in eng). J Infect Dis 180:1912–1920. https://doi.org/10.1086/315138

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Claro T, Widaa A, O’Seaghdha M, Miajlovic H, Foster TJ, O’Brien FJ, Kerrigan SW (2011) Staphylococcus aureus protein A binds to osteoblasts and triggers signals that weaken bone in osteomyelitis. PLoS ONE 6:e18748. https://doi.org/10.1371/journal.pone.0018748

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Wagner JM, Zollner H, Wallner C, Ismer B, Schira J, Abraham S, Harati K, Lehnhardt M, Behr B (2016) Surgical debridement is superior to sole antibiotic therapy in a novel murine posttraumatic osteomyelitis model. PLoS ONE 11:e0149389. https://doi.org/10.1371/journal.pone.0149389

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Wagner JM, Jaurich H, Wallner C, Abraham S, Becerikli M, Dadras M, Harati K, Duhan V, Khairnar V, Lehnhardt M, Behr B (2017) Diminished bone regeneration after debridement of posttraumatic osteomyelitis is accompanied by altered cytokine levels, elevated B cell activity and increased osteoclast activity (in eng). J Orthop Res. https://doi.org/10.1002/jor.23555

    Article  PubMed  Google Scholar 

  12. 12.

    Wagner JM, Reinkemeier F, Wallner C, Dadras M, Huber J, Schmidt SV, Drysch M, Dittfeld S, Jaurich H, Becerikli M, Becker K, Rauch N, Duhan V, Lehnhardt M, Behr B (2019) Adipose derived stromal cells (ASCs) are capable to restore bone regeneration after posttraumatic osteomyelitis and modulate B-cell response. Stem Cells Transl Med doi:DOI. https://doi.org/10.1002/sctm.18-0266

    Article  Google Scholar 

  13. 13.

    Wagner JM, Reinkemeier F, Dadras M, Wallner C, Huber J, Sogorski A, Sacher M, Schmidt S, Drysch M, Dittfeld S, Becerikli M, Becker K, Rauch N, Lehnhardt M, Behr B (2020) Local Wnt3a treatment restores bone regeneration in large osseous defects after surgical debridement of osteomyelitis (in eng). J Mol Med (Berl). https://doi.org/10.1007/s00109-020-01924-9

    Article  Google Scholar 

  14. 14.

    Bakker AD, Klein-Nulend J (2012) Osteoblast isolation from murine calvaria and long bones. Methods Mol Biol 816:19–29. https://doi.org/10.1007/978-1-61779-415-5_2

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Wallner C, Huber J, Drysch M, Schmidt SV, Wagner JM, Dadras M, Dittfeld S, Becerikli M, Jaurich H, Lehnhardt M, Behr B (2019) Activin receptor 2 antagonization impairs adipogenic and enhances osteogenic differentiation in mouse adipose-derived stem cells and mouse bone marrow-derived stem cells in vitro and in vivo (in eng). Stem Cells Dev 28:384–397. https://doi.org/10.1089/scd.2018.0155

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Wallner C, Abraham S, Wagner JM, Harati K, Ismer B, Kessler L, Zollner H, Lehnhardt M, Behr B (2016) Local application of isogenic adipose-derived stem cells restores bone healing capacity in a type 2 diabetes model (in eng). Stem Cells Transl Med 5:836–844. https://doi.org/10.5966/sctm.2015-0158

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    J E, S R, W R, (1999) Mechanisms of S aureus Invasion of cultured osteoblasts. Microb Pathog 26:317–323

    Article  Google Scholar 

  18. 18.

    Behr B, Ko SH, Wong VW, Gurtner GC, Longaker MT (2010) Stem cells. (in eng). Plast Reconstr Surg 126:1163–1171. https://doi.org/10.1097/PRS.0b013e3181ea42bb

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Chen Q, Hou T, Luo F, Wu X, Xie Z, Xu J (2014) Involvement of toll-like receptor 2 and pro-apoptotic signaling pathways in bone remodeling in osteomyelitis (in eng). Cell Physiol Biochem 34:1890–1900. https://doi.org/10.1159/000366387

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Sanchez CJ Jr, Ward CL, Romano DR, Hurtgen BJ, Hardy SK, Woodbury RL, Trevino AV, Rathbone CR, Wenke JC (2013) Staphylococcus aureus biofilms decrease osteoblast viability, inhibits osteogenic differentiation, and increases bone resorption in vitro (in eng). BMC Musculoskelet Disord 14:187. https://doi.org/10.1186/1471-2474-14-187

    Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Tang C-H, Kassem A, Lindholm C, Lerner UH (2016) Toll-Like receptor 2 stimulation of osteoblasts mediates S. Aureus induced bone resorption and osteoclastogenesis through enhanced rankl. PLoS ONE 11:e0156708. https://doi.org/10.1371/journal.pone.0156708

    CAS  Article  Google Scholar 

  22. 22.

    Somayaji SN, Huet YM, Gruber HE, Hudson MC (2010) UV-killed S. Aureus enhances adhesion and differentiation of osteoblasts on bone-associated biomaterials (in eng). J Biomed Mater Res A 95:574–579. https://doi.org/10.1002/jbm.a.32890

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Jin T, Lu Y, He QX, Wang H, Li BF, Zhu LY, Xu QY (2015) The role of microrNA, miR-24, and its target CHI3L1 in osteomyelitis caused by S. Aureus (in eng). J Cell Biochem 116:2804–2813. https://doi.org/10.1002/jcb.25225

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Tucker KA, Reilly SS, Leslie CS, Hudson MC (2000) Intracellular S. Aureus induces apoptosis in mouse osteoblasts (in eng). FEMS Microbiol Lett 186:151–156. https://doi.org/10.1111/j.1574-6968.2000.tb09096.x

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Young AB, Cooley ID, Chauhan VS, Marriott I (2011) Causative agents of osteomyelitis induce death domain-containing TNF-related apoptosis-inducing ligand receptor expression on osteoblasts (in eng). Bone 48:857–863. https://doi.org/10.1016/j.bone.2010.11.015

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Neumann J, Endermann T, Ehlers S, Reiling N (2009) Inverse relationship of TLR/NF-κB signalling and the Wnt/β-catenin pathway during inflammation: deciphering the role of Frizzled1 in M. tuberculosis infection. Cell Commun Signal 7:A52. https://doi.org/10.1186/1478-811X-7-S1-A52

    Article  PubMed Central  Google Scholar 

  27. 27.

    Neumann J, Schaale K, Farhat K, Endermann T, Ulmer AJ, Ehlers S, Reiling N (2010) Frizzled1 is a marker of inflammatory macrophages, and its ligand Wnt3a is involved in reprogramming Mycobacterium tuberculosis-infected macrophages. (in eng). FASEB J 24:4599–4612. https://doi.org/10.1096/fj.10-160994

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Widaa A, Claro T, Foster TJ, O’Brien FJ, Kerrigan SW (2012) Staphylococcus aureus protein A plays a critical role in mediating bone destruction and bone loss in osteomyelitis. PLoS ONE 7:e40586. https://doi.org/10.1371/journal.pone.0040586

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Glass DA 2nd, Bialek P, Ahn JD, Starbuck M, Patel MS, Clevers H, Taketo MM, Long F, McMahon AP, Lang RA, Karsenty G (2005) Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation (in eng). Dev Cell 8:751–764. https://doi.org/10.1016/j.devcel.2005.02.017

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Friedman MS, Oyserman SM, Hankenson KD (2009) Wnt11 promotes osteoblast maturation and mineralization through R-spondin 2 (in eng). J Biol Chem 284:14117–14125. https://doi.org/10.1074/jbc.M808337200

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Tresguerres FGF, Torres J, López-Quiles J, Hernández G, Vega JA, Tresguerres IF (2020) The osteocyte: a multifunctional cell within the bone (in eng). Ann Anat 227:151422. https://doi.org/10.1016/j.aanat.2019.151422

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Franz-Odendaal TA, Hall BK, Witten PE (2006) Buried alive: how osteoblasts become osteocytes (in eng). Dev Dyn 235:176–190. https://doi.org/10.1002/dvdy.20603

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Hanada R, Hanada T, Sigl V, Schramek D, Penninger JM (2011) RANKL/RANK-beyond bones (in eng). J Mol Med (Berl) 89:647–656. https://doi.org/10.1007/s00109-011-0749-z

    CAS  Article  Google Scholar 

  34. 34.

    Nakashima T, Hayashi M, Fukunaga T, Kurata K, Oh-Hora M, Feng JQ, Bonewald LF, Kodama T, Wutz A, Wagner EF, Penninger JM, Takayanagi H (2011) Evidence for osteocyte regulation of bone homeostasis through RANKL expression (in eng). Nat Med 17:1231–1234. https://doi.org/10.1038/nm.2452

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Bodine PV, Billiard J, Moran RA, Ponce-de-Leon H, McLarney S, Mangine A, Scrimo MJ, Bhat RA, Stauffer B, Green J, Stein GS, Lian JB, Komm BS (2005) The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis (in eng). J Cell Biochem 96:1212–1230. https://doi.org/10.1002/jcb.20599

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Yao W, Cheng Z, Shahnazari M, Dai W, Johnson ML, Lane NE (2010) Overexpression of secreted frizzled-related protein 1 inhibits bone formation and attenuates parathyroid hormone bone anabolic effects (in eng). J Bone Miner Res 25:190–199. https://doi.org/10.1359/jbmr.090719

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Häusler KD, Horwood NJ, Chuman Y, Fisher JL, Ellis J, Martin TJ, Rubin JS, Gillespie MT (2004) Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation (in eng). J Bone Miner Res 19:1873–1881. https://doi.org/10.1359/jbmr.040807

    Article  PubMed  Google Scholar 

  38. 38.

    Wang Y, Volloch V, Pindrus MA, Blasioli DJ, Chen J, Kaplan DL (2007) Murine osteoblasts regulate mesenchymal stem cells via WNT and cadherin pathways: mechanism depends on cell-cell contact mode (in eng). J Tissue Eng Regen Med 1:39–50. https://doi.org/10.1002/term.6

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Rinker TE, Hammoudi TM, Kemp ML, Lu H, Temenoff JS (2014) Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment (in eng). Integr Biol 6:324–337. https://doi.org/10.1039/c3ib40194d

    CAS  Article  Google Scholar 

  40. 40.

    Chung YS, Baylink DJ, Srivastava AK, Amaar Y, Tapia B, Kasukawa Y, Mohan S (2004) Effects of secreted frizzled-related protein 3 on osteoblasts in vitro (in eng). J Bone Miner Res 19:1395–1402. https://doi.org/10.1359/jbmr.040412

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Khamees N, Hill DJ, Kafienah W (2020) Mechanisms of interaction of S. Aureus with human mesenchymal stem cells and their differentiated phenotypes. bioRxiv. 14:184. https://doi.org/10.1101/2020.01.09.900373

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant of the DFG (Deutsche Forschungsgemeinschaft) (BE 4169/8-1).

Author information

Affiliations

Authors

Contributions

JW, YS, SD, and MB did all the experiments and BB, JW, YS, FR, MD, CW, JH, AS, MS, SD, MB, ML interpreted data and contributed to research design. All authors have read and accepted the final version of the manuscript.

Corresponding author

Correspondence to Johannes Maximilian Wagner.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interests.

Ethical approval

Isolation of murine osteoblasts was performed according to the guidelines of the National Institute of Health for the use of experimental animals and after approval by the German legislation. The protocol was approved by the LANUV (NRW, Germany; Permit-Number: 84–02.04.2014.A044).

Informed consent

Informed consent is not applicable for this study.

Additional information

Publisher's Note

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

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wagner, J.M., Steubing, Y., Dadras, M. et al. Wnt3a and ASCs are capable of restoring mineralization in staph aureus-infected primary murine osteoblasts. J Bone Miner Metab (2021). https://doi.org/10.1007/s00774-021-01269-4

Download citation

Keywords

  • Osteoblast
  • Osteomyelitis
  • Wnt3a
  • ASC