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Cell and Tissue Research

, Volume 368, Issue 3, pp 551–561 | Cite as

TSG-6 secreted by mesenchymal stem cells suppresses immune reactions influenced by BMP-2 through p38 and MEK mitogen-activated protein kinase pathway

  • Soyoun Um
  • Hui Young Kim
  • Joo-Hee Lee
  • In-Seok Song
  • Byoung Moo SeoEmail author
Regular Article

Abstract

Bone morphogenetic protein 2 (BMP-2) has a critical function in bone and cartilage development and in repairing damaged organs and tissue. However, clinical use of BMP-2 at doses of 0.5–1 mg/ml for orthopedics has been associated with severe postoperative swelling requiring emergency surgical intervention. We determined whether a high concentration of BMP-2 induces inflammatory responses in macrophages and the suppression of osteogenesis in hMSCs. We obtained human periodontal ligament stem cells and bone marrow stem cells from the maxilla, i.e., human mesenchymal stem cells (hMSCs), from the periodontal ligament of extracted third molar teeth and from the bone marrow of the maxilla, respectively. Osteogenic differentiation was measured by alkaline phosphatase activity and alizarin red S staining. Proteins were assessed by flow cytometry, enzyme-linked immunosorbent assay, Western blot and immunocytochemistry. Changes of gene expression were measured by reverse transcription plus the polymerase chain reaction (RT-PCR) and real-time PCR. A high BMP-2 concentration inhibited the early stages of osteogenesis in hMSCs. Co-culturing THP-1 cells (human monocytic cells) with hMSCs reduced the late stages of osteogenesis compared with those seen in hMSCs alone. In addition, high-dose BMP-2 induced the expression of inflammatory cytokines in THP-1 cells and the expression of the anti-inflammatory cytokine tumor-necrosis-factor-α-inducible gene 6 protein (TSG-6) in hMSCs. Consistent with the anti-inflammatory effects of hMSCs when co-cultured with THP-1 cells, interleukin-1β expression was downregulated by TSG-6 treatment of THP-1 cells. Our findings suggest that a high BMP-2 concentration triggers inflammation that causes inflammatory cytokine release from THP-1 cells, leading to the suppression of osteogenesis, whereas TSG-6 secreted by hMSCs suppresses inflammatory reactions through p38 and ERK in the mitogen-activated protein kinase pathway.

Keywords

Periodontal ligament (PDL) Stem cells Regeneration Osteogenesis Inflammation 

Notes

Acknowledgements

This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI12C0763). BMP-2 was kindly donated by the Daewoong Pharmaceutical Company without any conditions.

Supplementary material

441_2017_2581_MOESM1_ESM.docx (14 kb)
Table S1 (DOCX 14 kb)
441_2017_2581_Fig7_ESM.gif (154 kb)
Fig. S1

High concentrations of BMP-2 trigger the infiltration of immune cells from blood vessels. a Collagen sponges (8 mm in diameter, 1 mm in thickness) as carriers were transplanted with 1, 5, 10, and 20 μg/ml BMP-2 onto the dorsum of mouse. b After 24 h, inflammatory cells were identified on the swollen soft tissue near the implanted collagen sponge with BMP-2 in hematoxylin and eosin staining. c To detect infiltrated activated macrophages, immunohistochemistry was performed via staining with F4/80 antibody (Abcam 100790, 1:100). Red arrows indicate activated macrophages. (GIF 153 kb)

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High resolution image (TIF 8993 kb)
441_2017_2581_Fig8_ESM.gif (59 kb)
Fig. S2

Morphology of THP-1 cells. Without PMA stimulation, THP-1 cells alone cannot be stimulated with BMP-2 treatment. (GIF 59 kb)

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High resolution image (TIF 9143 kb)
441_2017_2581_Fig9_ESM.gif (76 kb)
Fig. S3

Flow cytometry data of THP-1 cells stimulated with BMP-2. After 3 days of 50 nM PMA stimulation, THP-1 cells were treated with BMP-2 for 24 h. THP-1 cells double-stained with CD11b and CD14 were increased in a BMP-2 dose dependent manner. Each experiment was repeated three times. (GIF 75 kb)

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High resolution image (TIF 11205 kb)
441_2017_2581_Fig10_ESM.gif (95 kb)
Fig. S4

Antibody array of THP-1 cells with or without PDLSC/BMSCs. a Array map (POS positive control spot, NEG negative control spot, BLANK blank spot). b THP-1 cells were activated with 50 nM PMA for 3 days and triggered with 5000 ng/ml BMP-2 for 24 hours. Inflammatory cytokine expression was enhanced upon BMP-2 induction. Co-culturing MSCs and THP-1 cells resulted in lower levels of inflammatory cytokines than in THP-1 cells alone (arrows anti-inflammatory cytokines, such as IL-10 and TGF-β1). (GIF 95 kb)

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High resolution image (TIF 9272 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Soyoun Um
    • 1
  • Hui Young Kim
    • 2
  • Joo-Hee Lee
    • 2
  • In-Seok Song
    • 3
  • Byoung Moo Seo
    • 2
    • 4
    Email author
  1. 1.Biotooth Engineering Laboratory, Dental Research Institute, Dental Regenerative Biotechnology, Department of Dental Science, School of DentistrySeoul National UniversitySeoulRepublic of Korea
  2. 2.Biotooth Engineering Laboratory, Department of Oral and Maxillofacial Surgery and Craniomaxillofacial Life Science, Dental Research Institute, School of DentistrySeoul National UniversitySeoulRepublic of Korea
  3. 3.Division of Oral and Maxillofacial Surgery, Department of DentistryKorea University Anam HospitalSeoulRepublic of Korea
  4. 4.Department of Oral and Maxillofacial SurgerySeoul National UniversitySeoulRepublic of Korea

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