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


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.


Periodontal ligament (PDL) Stem cells Regeneration Osteogenesis Inflammation 



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)
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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)


  1. Akintoye SO, Lam T, Shi S, Brahim J, Collins MT, Robey PG (2006) Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals. Bone 38:758–768CrossRefPubMedGoogle Scholar
  2. Boyne PJ, Lilly LC, Marx RE, Moy PK, Nevins M, Spagnoli DB, Triplett RG (2005) De novo bone induction by recombinant human bone morphogenetic protein-2 (rhBMP-2) in maxillary sinus floor augmentation. J Oral Maxillofac Surg 63:1693–1707CrossRefPubMedGoogle Scholar
  3. Castro-Manrreza ME, Mayani H, Monroy-Garcia A, Flores-Figueroa E, Chavez-Rueda K, Legorreta-Haquet V, Santiago-Osorio E, Montesinos JJ (2014) Human mesenchymal stromal cells from adult and neonatal sources: a comparative in vitro analysis of their immunosuppressive properties against T cells. Stem Cells Dev 23:1217–1232CrossRefPubMedPubMedCentralGoogle Scholar
  4. Choi H, Lee RH, Bazhanov N, Oh JY, Prockop DJ (2011) Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-kappaB signaling in resident macrophages. Blood 118:330–338CrossRefPubMedPubMedCentralGoogle Scholar
  5. Fiorellini JP, Howell TH, Cochran D, Malmquist J, Lilly LC, Spagnoli D, Toljanic J, Jones A, Nevins M (2005) Randomized study evaluating recombinant human bone morphogenetic protein-2 for extraction socket augmentation. J Periodontol 76:605–613CrossRefPubMedGoogle Scholar
  6. Gibon E, Lu L, Goodman SB (2016) Aging, inflammation, stem cells, and bone healing. Stem Cell Res Ther 7:44CrossRefPubMedPubMedCentralGoogle Scholar
  7. Guan J, Zhang J, Zhu Z, Niu X, Guo S, Wang Y, Zhang C (2015) Bone morphogenetic protein 2 gene transduction enhances the osteogenic potential of human urine-derived stem cells. Stem Cell Res Ther 6:5CrossRefPubMedPubMedCentralGoogle Scholar
  8. Haynes DR, Crotti TN, Loric M, Bain GI, Atkins GJ, Findlay DM (2001) Osteoprotegerin and receptor activator of nuclear factor kappaB ligand (RANKL) regulate osteoclast formation by cells in the human rheumatoid arthritic joint. Rheumatology (Oxford) 40:623–630CrossRefGoogle Scholar
  9. Hong JH, Lee GT, Lee JH, Kwon SJ, Park SH, Kim SJ, Kim IY (2009) Effect of bone morphogenetic protein-6 on macrophages. Immunology 128:e442–e450CrossRefPubMedPubMedCentralGoogle Scholar
  10. Huang GT, Gronthos S, Shi S (2009) Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 88:792–806CrossRefPubMedPubMedCentralGoogle Scholar
  11. Huang RL, Yuan Y, Tu J, Zou GM, Li Q (2014a) Opposing TNF-alpha/IL-1beta- and BMP-2-activated MAPK signaling pathways converge on Runx2 to regulate BMP-2-induced osteoblastic differentiation. Cell Death Dis 5:e1187CrossRefPubMedPubMedCentralGoogle Scholar
  12. Huang RL, Yuan Y, Zou GM, Liu G, Tu J, Li Q (2014b) LPS-stimulated inflammatory environment inhibits BMP-2-induced osteoblastic differentiation through crosstalk between TLR4/MyD88/NF-kappaB and BMP/Smad signaling. Stem Cells Dev 23:277–289CrossRefPubMedGoogle Scholar
  13. Hume DA, Ross IL, Himes SR, Sasmono RT, Wells CA, Ravasi T (2002) The mononuclear phagocyte system revisited. J Leukoc Biol 72:621–627PubMedGoogle Scholar
  14. Kim BC, Bae H, Kwon IK, Lee EJ, Park JH, Khademhosseini A, Hwang YS (2012) Osteoblastic/cementoblastic and neural differentiation of dental stem cells and their applications to tissue engineering and regenerative medicine. Tissue Eng Part B Rev 18:235–244CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kim J, Hematti P (2009) Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages. Exp Hematol 37:1445–1453CrossRefPubMedPubMedCentralGoogle Scholar
  16. Krötzsch E, Salgado RM, Caba D, Lichtinger A, Padilla L, Di Silvio M (2005) Alkaline phosphatase activity is related to acute inflammation and collagen turnover during acute and chronic wound healing. Wound Repair Regen 13:A28–A48CrossRefGoogle Scholar
  17. Lee DE, Ayoub N, Agrawal DK (2016) Mesenchymal stem cells and cutaneous wound healing: novel methods to increase cell delivery and therapeutic efficacy. Stem Cell Res Ther 7:37CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lee GT, Kwon SJ, Lee JH, Jeon SS, Jang KT, Choi HY, Lee HM, Kim WJ, Kim SJ, Kim IY (2010) Induction of interleukin-6 expression by bone morphogenetic protein-6 in macrophages requires both SMAD and p38 signaling pathways. J Biol Chem 285:39401–39408CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lee KB, Murray SS, Taghavi CE, Song KJ, Brochmann EJ, Johnson JS, Keorochana G, Liao JC, Wang JC (2011a) Bone morphogenetic protein-binding peptide reduces the inflammatory response to recombinant human bone morphogenetic protein-2 and recombinant human bone morphogenetic protein-7 in a rodent model of soft-tissue inflammation. Spine J 11:568–576CrossRefPubMedGoogle Scholar
  20. Lee KB, Taghavi CE, Song KJ, Sintuu C, Yoo JH, Keorochana G, Tzeng ST, Fei Z, Liao JC, Wang JC (2011b) Inflammatory characteristics of rhBMP-2 in vitro and in an in vivo rodent model. Spine 36:E149–E154CrossRefPubMedGoogle Scholar
  21. Lee KB, Taghavi CE, Murray SS, Song KJ, Keorochana G, Wang JC (2012) BMP induced inflammation: a comparison of rhBMP-7 and rhBMP-2. J Orthop Res 30:1985–1994CrossRefPubMedGoogle Scholar
  22. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, Semprun-Prieto L, Delafontaine P, Prockop DJ (2009a) Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 5:54–63CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lee RH, Seo MJ, Pulin AA, Gregory CA, Ylostalo J, Prockop DJ (2009b) The CD34-like protein PODXL and alpha6-integrin (CD49f) identify early progenitor MSCs with increased clonogenicity and migration to infarcted heart in mice. Blood 113:816–826CrossRefPubMedPubMedCentralGoogle Scholar
  24. Lee RH, Yu JM, Foskett AM, Peltier G, Reneau JC, Bazhanov N, Oh JY, Prockop DJ (2014) TSG-6 as a biomarker to predict efficacy of human mesenchymal stem/progenitor cells (hMSCs) in modulating sterile inflammation in vivo. Proc Natl Acad Sci U S A 111:16766–16771CrossRefPubMedPubMedCentralGoogle Scholar
  25. Liu M, Zeng X, Wang J, Fu Z, Wang J, Liu M, Ren D, Yu B, Zheng L, Hu X, Shi W, Xu J (2016) Immunomodulation by mesenchymal stem cells in treating human autoimmune disease-associated lung fibrosis. Stem Cell Res Ther 7:63CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ma S, Xie N, Li W, Yuan B, Shi Y, Wang Y (2014) Immunobiology of mesenchymal stem cells. Cell Death Differ 21:216–225CrossRefPubMedGoogle Scholar
  27. Mahoney DJ, Mikecz K, Ali T, Mabilleau G, Benayahu D, Plaas A, Milner CM, Day AJ, Sabokbar A (2008) TSG-6 regulates bone remodeling through inhibition of osteoblastogenesis and osteoclast activation. J Biol Chem 283:25952–25962CrossRefPubMedPubMedCentralGoogle Scholar
  28. Manning CN, Martel C, Sakiyama-Elbert SE, Silva MJ, Shah S, Gelberman RH, Thomopoulos S (2015) Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro. Stem Cell Res Ther 6:74CrossRefPubMedPubMedCentralGoogle Scholar
  29. Manolagas SC, Jilka RL (1995) Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. N Engl J Med 332:305–311CrossRefPubMedGoogle Scholar
  30. Mundy GR (2007) Osteoporosis and inflammation. Nutr Rev 65:S147–S151CrossRefPubMedGoogle Scholar
  31. Nazarov C, Lo Surdo J, Bauer SR, Wei CH (2013) Assessment of immunosuppressive activity of human mesenchymal stem cells using murine antigen specific CD4 and CD8 T cells in vitro. Stem Cell Res Ther 4:128CrossRefPubMedPubMedCentralGoogle Scholar
  32. Nemeth K, Leelahavanichkul A, Yuen PS, Mayer B, Parmelee A, Doi K, Robey PG, Leelahavanichkul K, Koller BH, Brown JM, Hu X, Jelinek I, Star RA, Mezey E (2009) Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 15:42–49CrossRefPubMedGoogle Scholar
  33. Perri B, Cooper M, Lauryssen C, Anand N (2007) Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion: a case study. Spine J 7:235–239CrossRefPubMedGoogle Scholar
  34. Pirraco RP, Reis RL, Marques AP (2013) Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs. J Tissue Eng Regen Med 7:392–400CrossRefPubMedGoogle Scholar
  35. Prockop DJ, Oh JY (2012) Medical therapies with adult stem/progenitor cells (MSCs): a backward journey from dramatic results in vivo to the cellular and molecular explanations. J Cell Biochem 113:1460–1469PubMedPubMedCentralGoogle Scholar
  36. Redlich K, Smolen JS (2012) Inflammatory bone loss: pathogenesis and therapeutic intervention. Nat Rev Drug Discov 11:234–250CrossRefPubMedGoogle Scholar
  37. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364:149–155CrossRefPubMedGoogle Scholar
  38. Shead EF, Haworth CS, Barker H, Bilton D, Compston JE (2010) Osteoclast function, bone turnover and inflammatory cytokines during infective exacerbations of cystic fibrosis. J Cyst Fibros 9:93–98CrossRefPubMedGoogle Scholar
  39. Shen J, James AW, Zara JN, Asatrian G, Khadarian K, Zhang JB, Ho S, Kim HJ, Ting K, Soo C (2013) BMP2-induced inflammation can be suppressed by the osteoinductive growth factor NELL-1. Tissue Eng Part A 19:2390–2401CrossRefPubMedPubMedCentralGoogle Scholar
  40. Shields LB, Raque GH, Glassman SD, Campbell M, Vitaz T, Harpring J, Shields CB (2006) Adverse effects associated with high-dose recombinant human bone morphogenetic protein-2 use in anterior cervical spine fusion. Spine 31:542–547CrossRefPubMedGoogle Scholar
  41. Simoes Sato AY, Bub GL, Campos AH (2014) BMP-2 and -4 produced by vascular smooth muscle cells from atherosclerotic lesions induce monocyte chemotaxis through direct BMPRII activation. Atherosclerosis 235:45–55CrossRefPubMedGoogle Scholar
  42. Smucker JD, Rhee JM, Singh K, Yoon ST, Heller JG (2006) Increased swelling complications associated with off-label usage of rhBMP-2 in the anterior cervical spine. Spine 31:2813–2819CrossRefPubMedGoogle Scholar
  43. Sullivan CB, Porter RM, Evans CH, Ritter T, Shaw G, Barry F, Murphy JM (2014) TNFalpha and IL-1beta influence the differentiation and migration of murine MSCs independently of the NF-kappaB pathway. Stem Cell Res Ther 5:104CrossRefPubMedPubMedCentralGoogle Scholar
  44. Swiontkowski MF, Aro HT, Donell S, Esterhai JL, Goulet J, Jones A, Kregor PJ, Nordsletten L, Paiement G, Patel A (2006) Recombinant human bone morphogenetic protein-2 in open tibial fractures. A subgroup analysis of data combined from two prospective randomized studies. J Bone Joint Surg Am 88:1258–1265PubMedGoogle Scholar
  45. Takabayashi H, Shinohara M, Mao M, Phaosawasdi P, El-Zaatari M, Zhang M, Ji T, Eaton KA, Dang D, Kao J, Todisco A (2014) Anti-inflammatory activity of bone morphogenetic protein signaling pathways in stomachs of mice. Gastroenterology 147:e397CrossRefGoogle Scholar
  46. Talati M, West J, Zaynagetdinov R, Hong CC, Han W, Blackwell T, Robinson L, Blackwell TS, Lane K (2014) BMP pathway regulation of and by macrophages. PLoS One 9:e94119CrossRefPubMedPubMedCentralGoogle Scholar
  47. Teitelbaum SL (2000) Bone resorption by osteoclasts. Science 289:1504–1508CrossRefPubMedGoogle Scholar
  48. Toth JM, Boden SD, Burkus JK, Badura JM, Peckham SM, McKay WF (2009) Short-term osteoclastic activity induced by locally high concentrations of recombinant human bone morphogenetic protein-2 in a cancellous bone environment. Spine 34:539–550CrossRefPubMedGoogle Scholar
  49. Tumialan LM, Pan J, Rodts GE, Mummaneni PV (2008) The safety and efficacy of anterior cervical discectomy and fusion with polyetheretherketone spacer and recombinant human bone morphogenetic protein-2: a review of 200 patients. J Neurosurg Spine 8:529–535CrossRefPubMedGoogle Scholar
  50. Vaidya R, Carp J, Sethi A, Bartol S, Craig J, Les CM (2007) Complications of anterior cervical discectomy and fusion using recombinant human bone morphogenetic protein-2. Eur Spine J 16:1257–1265CrossRefPubMedPubMedCentralGoogle Scholar
  51. Vasandan AB, Shankar SR, Prasad P, Sowmya Jahnavi V, Bhonde RR, Jyothi Prasanna S (2014) Functional differences in mesenchymal stromal cells from human dental pulp and periodontal ligament. J Cell Mol Med 18:344–354CrossRefPubMedPubMedCentralGoogle Scholar
  52. Wang N, Shao Y, Mei Y, Zhang L, Li Q, Li D, Shi S, Hong Q, Lin H, Chen X (2012) Novel mechanism for mesenchymal stem cells in attenuating peritoneal adhesion: accumulating in the lung and secreting tumor necrosis factor alpha-stimulating gene-6. Stem Cell Res Ther 3:51CrossRefPubMedPubMedCentralGoogle Scholar
  53. Wei S, Cai X, Huang J, Xu F, Liu X, Wang Q (2012) Recombinant human BMP-2 for the treatment of open tibial fractures. Orthopedics 35:e847–e854CrossRefPubMedGoogle Scholar
  54. Zara JN, Siu RK, Zhang X, Shen J, Ngo R, Lee M, Li W, Chiang M, Chung J, Kwak J, Wu BM, Ting K, Soo C (2011) High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo. Tissue Eng Part A 17:1389–1399CrossRefPubMedPubMedCentralGoogle Scholar

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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