Methylsulfonylmethane increases osteogenesis and regulates the mineralization of the matrix by transglutaminase 2 in SHED cells

Autoři: Hanan Aljohani aff001;  Linda T. Senbanjo aff001;  Meenakshi A. Chellaiah aff001
Působiště autorů: Department of Oncology and Diagnostic Sciences, University of Maryland School of Dentistry, Baltimore, MD, United States of America aff001;  Department of Oral Medicine and Diagnostics Sciences, King Saud University School of Dentistry, Riyadh, KSA aff002
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM.

Klíčová slova:

Cell differentiation – Cell staining – Collagens – Gene expression – Immunoblotting – Immunoprecipitation – Mesenchymal stem cells – Osteoblast differentiation


1. Papaccio F, Paino F, Regad T, Papaccio G, Desiderio V, Tirino V. Concise Review: Cancer Cells, Cancer Stem Cells, and Mesenchymal Stem Cells: Influence in Cancer Development. Stem Cells Transl Med. 2017;2115–25. doi: 10.1002/sctm.17-0138 29072369

2. Huang GT-J, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009 Sep;792–806. doi: 10.1177/0022034509340867 19767575

3. Bianco P, Robey PG. Stem cells in tissue engineering. Nature. 2001 Nov 1;118–21.

4. Bluteau G, Luder HU, De Bari C, Mitsiadis TA. Stem cells for tooth engineering. Eur Cells Mater. 2008;1–9.

5. Carinci F, Papaccio G, Laino G, Palmieri A, Brunelli G, D’Aquino R, et al. Comparison Between Genetic Portraits of Osteoblasts Derived From Primary Cultures and Osteoblasts Obtained From Human Pulpar Stem Cells. J Craniofac Surg. 2008 May;616–25. doi: 10.1097/SCS.0b013e31816aabc8 18520373

6. Laino G, D’Aquino R, Graziano A, Lanza V, Carinci F, Naro F, et al. A New Population of Human Adult Dental Pulp Stem Cells: A Useful Source of Living Autologous Fibrous Bone Tissue (LAB). J Bone Miner Res. 2005 Aug;1394–402. doi: 10.1359/JBMR.050325 16007337

7. Lima RL, Holanda-Afonso RC, Moura-Neto V, Bolognese AM, DosSantos MF, Souza MM. Human dental follicle cells express embryonic, mesenchymal and neural stem cells markers. Arch Oral Biol. 2017 Jan;121–8.

8. Vishwanath VR, Nadig RR, Nadig R, Prasanna JS, Karthik J, Pai VS. Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study. J Conserv Dent. 2013 Sep;423–8. doi: 10.4103/0972-0707.117509 24082571

9. Gosau M, Götz W, Felthaus O, Ettl T, Jäger A, Morsczeck C. Comparison of the differentiation potential of neural crest derived progenitor cells from apical papilla (dNC-PCs) and stem cells from exfoliated deciduous teeth (SHED) into mineralising cells. Arch Oral Biol. 2013;

10. Winning L, El Karim IA, Lundy FT. A Comparative Analysis of the Osteogenic Potential of Dental Mesenchymal Stem Cells. Stem Cells Dev. 2019 Aug 1;1050–8. doi: 10.1089/scd.2019.0023 31169063

11. Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;5807–12. doi: 10.1073/pnas.0937635100 12716973

12. Kashyap R. SHED—Basic Structure for Stem Cell Research. J Clin DIAGNOSTIC Res. 2015 Mar;ZE07–9.

13. Gazarian KG, Ramírez-García LR. Human Deciduous Teeth Stem Cells (SHED) Display Neural Crest Signature Characters. 2017;

14. D’Aquino R, Papaccio G, Laino G, Graziano A. Dental pulp stem cells: A promising tool for bone regeneration. Stem Cell Rev. 2008;21–6. doi: 10.1007/s12015-008-9013-5 18300003

15. Leyendecker Junior A, Gomes Pinheiro CC, Lazzaretti Fernandes T, Franco Bueno D. The use of human dental pulp stem cells for in vivo bone tissue engineering: A systematic review. J Tissue Eng. 2018 Jan 17;2041731417752766.

16. Butawan M, Benjamin RL, Bloomer RJ. Methylsulfonylmethane: Applications and safety of a novel dietary supplement. Nutrients. 2017 Mar 16;1–21.

17. Kim B-R, Ba T, Nguyen L, Min Y-K, Lee B-T, Nguyen TBL, et al. In vitro and in vivo studies of BMP-2-loaded PCL-gelatin-BCP electrospun scaffolds. Tissue Eng Part A. 2014 Dec;3279–89. doi: 10.1089/ten.TEA.2014.0081 24935525

18. Joung YH, Lim EJ, Darvin P, Chung SC, Jang JW, Do Park K, et al. MSM Enhances GH Signaling via the Jak2/STAT5b Pathway in Osteoblast-Like Cells and Osteoblast Differentiation through the Activation of STAT5b in MSCs. PLoS One. 2012;

19. Kim DN, Joung YH, Darvin P, Kang DY, Sp N, Byun HJ, et al. Methylsulfonylmethane enhances BMP‑2‑induced osteoblast differentiation in mesenchymal stem cells. Mol Med Rep. 2016 Jul;460–6.

20. Mohammadi S, Najafi M, Hamzeiy H, Maleki-Dizaji N, Pezeshkian M, Sadeghi-Bazargani H, et al. Protective Effects of Methylsulfonylmethane on Hemodynamics and Oxidative Stress in Monocrotaline-Induced Pulmonary Hypertensive Rats. Adv Pharmacol Sci. 2012;

21. Lim EJ, Hong DY, Park JH, Joung YH, Darvin P, Kim SY, et al. Methylsulfonylmethane suppresses breast cancer growth by down-regulating STAT3 and STAT5b pathways. Li J, editor. PLoS One. 2012 Apr 2;e33361. doi: 10.1371/journal.pone.0033361 22485142

22. Kaartinen MT, El-Maadawy S, Räsänen NH, McKee MD. Tissue Transglutaminase and Its Substrates in Bone. J Bone Miner Res. 2002 Dec 1;2161–73. doi: 10.1359/jbmr.2002.17.12.2161 12469910

23. Mousa A, Cui C, Song A, Myneni VD, Sun H, Li JJ, et al. Transglutaminases factor XIII-A and TG2 regulate resorption, adipogenesis and plasma fibronectin homeostasis in bone and bone marrow. Cell Death Differ. 2017 May 7;844–54. doi: 10.1038/cdd.2017.21 28387755

24. Lorand L, Dailey JE, Turner PM. Fibronectin as a carrier for the transglutaminase from human erythrocytes [Internet]. Vol. 85, Proc. Nati. Acad. Sci. USA. 1988.

25. Aeschlimann D, Thomazy V. Protein Crosslinking in Assembly and Remodelling of Extracellular Matrices: The Role of Transglutaminases. Connect Tissue Res. 2000 Jan 6;1–27. doi: 10.3109/03008200009005638 10826705

26. Lee CS, Park HH. Structural aspects of transglutaminase 2: functional, structural, and regulatory diversity. Apoptosis. 2017 Sep 4;1057–68. doi: 10.1007/s10495-017-1396-9 28677093

27. Bento LW, Zhang Z, Imai A, Nör F, Dong Z, Shi S, et al. Endothelial differentiation of SHED requires MEK1/ERK signaling. J Dent Res. 2013;51–7. doi: 10.1177/0022034512466263 23114032

28. Krebsbach PH, Robey PG. Dental and skeletal stem cells: potential cellular therapeutics for craniofacial regeneration. J Dent Educ. 2002 Jun;766–73. 12117099

29. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000 Dec 5;13625–30. doi: 10.1073/pnas.240309797 11087820

30. Wang Y, Papagerakis S, Faulk D, Badylak SF, Zhao Y, Ge L, et al. Extracellular Matrix Membrane Induces Cementoblastic/Osteogenic Properties of Human Periodontal Ligament Stem Cells. Front Physiol. 2018;942. doi: 10.3389/fphys.2018.00942 30072915

31. Gupta A, Cao W, Chellaiah MA. Integrin αvβ3 and CD44 pathways in metastatic prostate cancer cells support osteoclastogenesis via a Runx2/Smad 5/receptor activator of NF-κB ligand signaling axis. Mol Cancer. 2012 Sep 11;66. doi: 10.1186/1476-4598-11-66 22966907

32. Senbanjo LT, AlJohani H, Majumdar S, Chellaiah MA. Characterization of CD44 intracellular domain interaction with RUNX2 in PC3 human prostate cancer cells. Cell Commun Signal. 2019 Jul 22;80. doi: 10.1186/s12964-019-0395-6 31331331

33. Chellaiah M, Fitzgerald C, Alvarez U, Hruska K. c-Src is required for stimulation of gelsolin-associated phosphatidylinositol 3-kinase. J Biol Chem. 1998 May 8;11908–16. doi: 10.1074/jbc.273.19.11908 9565618

34. Chellaiah MA, Schaller MD. Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: Comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro. J Cell Physiol. 2009 Aug 1;382–93.

35. Chellaiah M, Hruska K. Osteopontin stimulates gelsolin-associated phosphoinositide levels and phosphatidylinositol triphosphate-hydroxyl kinase. Mol Biol Cell. 1996 May;743–53. doi: 10.1091/mbc.7.5.743 8744948

36. Chellaiah MA, Kizer N, Biswas R, Alvarez U, Strauss-Schoenberger J, Rifas L, et al. Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression. Yamamoto KR, editor. Mol Biol Cell. 2003 Jan;173–89. doi: 10.1091/mbc.E02-06-0354 12529435

37. Majumdar S, Wadajkar AS, Aljohani H, Reynolds MA, Kim AJ, Chellaiah M. Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro. Int J Cell Biol. 2019 May 5;6943986. doi: 10.1155/2019/6943986 31191656

38. Chellaiah MA, Majumdar S, Aljohani H. Peptidomimetic inhibitors of L-plastin reduce the resorptive activity of osteoclast but not the bone forming activity of osteoblasts in vitro. Reddy S V, editor. PLoS One. 2018 Sep 24;e0204209. doi: 10.1371/journal.pone.0204209 30248139

39. Goto T, Kajiwara H, Yoshinari M, Fukuhara E, Kobayashi S, Tanaka T. In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue. Biomaterials. 2003 Oct 1;3885–92. doi: 10.1016/s0142-9612(03)00258-8 12834583

40. Wang Y-H, Liu Y, Maye P, Rowe DW. Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes. Biotechnol Prog. 2006;1697–701. doi: 10.1021/bp060274b 17137320

41. Chellaiah M, Fitzgerald C, Filardo EJ, Cheresh DA, Hruska KA. Osteopontin activation of c-src in human melanoma cells requires the cytoplasmic domain of the integrin alpha v-subunit. Endocrinology. 1996 Jun;2432–40. doi: 10.1210/endo.137.6.8641196 8641196

42. Samanna V, Wei H, Ego-Osuala D, Chellaiah MA. Alpha-V-dependent outside-in signaling is required for the regulation of CD44 surface expression, MMP-2 secretion, and cell migration by osteopontin in human melanoma cells. Exp Cell Res. 2006 Jul 15;2214–30. doi: 10.1016/j.yexcr.2006.03.022 16631740

43. Tang N, Song W, Luo J, Luo X, Chen J, Sharff KA, et al. BMP‐9‐induced osteogenic differentiation of mesenchymal progenitors requires functional canonical Wnt/β‐catenin signalling. J Cell Mol Med. 2009;2448. doi: 10.1111/j.1582-4934.2008.00569.x 19175684

44. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell. 2002 Jan 11;17–29.

45. Achyuthan KE, Rowland TC, Birckbichler PJ, Lee KN, Bishop PD, Achyuthan AM. Hierarchies in the binding of human factor XIII, factor XIIIa, and endothelial cell transglutaminase to human plasma fibrinogen, fibrin, and fibronectin. Mol Cell Biochem. 1996 Sep 6;43–9.

46. Aeschlimannsg D, Paulssons M, Mannll K. Identification of Gln726 in Nidogen as the Amine Acceptor in Transglutaminase-catalyzed Cross-linking of Laminin-Nidogen Complexes*. J Biol Chem. 1992;11316–21. 1350783

47. Lynch GW, Slayter HS, Miller BE, McDonagh J. Characterization of thrombospondin as a substrate for factor XIII transglutaminase. J Biol Chem. 1987 Feb 5;1772–8. 2879842

48. Lee J, Condello S, Yakubov B, Emerson R, Caperell-Grant A, Hitomi K, et al. Tissue Transglutaminase Mediated Tumor-Stroma Interaction Promotes Pancreatic Cancer Progression. Clin Cancer Res. 2015 Oct 1;4482–93. doi: 10.1158/1078-0432.CCR-15-0226 26041746

49. Yin X, Chen Z, Liu Z, Song C. Tissue transglutaminase (TG2) activity regulates osteoblast differentiation and mineralization in the SAOS-2 cell line. Brazilian J Med Biol Res = Rev Bras Pesqui medicas e Biol. 2012 Aug;693–700.

50. Wozniak M, Fausto A, Carron CP, Meyer DM, Hruska KA. Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of αVβ3-Integrin Expression. J Bone Miner Res. 2000 Sep 1;1731–45. doi: 10.1359/jbmr.2000.15.9.1731 10976993

51. An J, Leeuwenburgh S, Wolke J, Jansen J. Mineralization processes in hard tissue: Bone. In: Biomineralization and Biomaterials. Woodhead Publishing; 2016. p. 129–46.

52. Xu J, Li Z, Hou Y, Fang W. Potential mechanisms underlying the Runx2 induced osteogenesis of bone marrow mesenchymal stem cells. Am J Transl Res. 2015;2527–35. 26885254

53. La Noce M, Mele L, Laino L, Iolascon G, Pieretti G, Papaccio G, et al. Cytoplasmic Interactions between the Glucocorticoid Receptor and HDAC2 Regulate Osteocalcin Expression in VPA-Treated MSCs. Cells. 2019 Mar 5;217.

54. Aubin JE. Regulation of osteoblast formation and function. Rev Endocr Metab Disord. 2001 Jan;81–94. doi: 10.1023/a:1010011209064 11704982

55. Štefková K, Procházková J, Pacherník J. Alkaline phosphatase in stem cells. Stem Cells Int. 2015 Feb 12;628368. doi: 10.1155/2015/628368 25767512

56. Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992 Jun 3;683–92. doi: 10.1002/jbmr.5650070613 1414487

57. Hoemann CD, El-Gabalawy H, McKee MD. In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization. Pathol Biol (Paris). 2009 Jun;318–23.

58. Huang Z, Nelson ER, Smith RL, Goodman SB. The Sequential Expression Profiles of Growth Factors from Osteroprogenitors to Osteoblasts In Vitro. Tissue Eng. 2007 Sep;2311–20. doi: 10.1089/ten.2006.0423 17523879

59. Paino F, La Noce M, Giuliani A, De Rosa A, Mazzoni S, Laino L, et al. Human DPSCs fabricate vascularized woven bone tissue: a new tool in bone tissue engineering. Clin Sci. 2017 Apr 25;699–713. doi: 10.1042/CS20170047 28209631

60. Telci D, Collighan RJ, Basaga H, Griffin M. Increased TG2 expression can result in induction of transforming growth factor beta1, causing increased synthesis and deposition of matrix proteins, which can be regulated by nitric oxide. J Biol Chem. 2009 Oct 23;29547–58. doi: 10.1074/jbc.M109.041806 19657147

61. Lesort M, Attanavanich K, Zhang J, Johnson GVW. Distinct Nuclear Localization and Activity of Tissue Transglutaminase. J Biol Chem. 1998 May 15;11991–4. doi: 10.1074/jbc.273.20.11991 9575137

62. Singh US, Erickson JW, Cerione RA. Identification and biochemical characterization of an 80 kilodalton GTP-binding/transglutaminase from rabbit liver nuclei. Biochemistry. 1995 Dec 5;15863–71. doi: 10.1021/bi00048a032 7495818

63. Schorn L, Sproll C, Ommerborn M, Naujoks C, Kübler NR, Depprich R. Vertical bone regeneration using rhBMP-2 and VEGF. Head Face Med. 2017 Jun 7;11. doi: 10.1186/s13005-017-0146-0 28592312

64. Maiorana C, Poli PP, Deflorian M, Testori T, Mandelli F, Nagursky H, et al. Alveolar socket preservation with demineralised bovine bone mineral and a collagen matrix. J Periodontal Implant Sci. 2017 Aug;194–210. doi: 10.5051/jpis.2017.47.4.194 28861284

65. Wood RA, Mealey BL. Histological Comparison of Healing Following Tooth Extraction With Ridge Preservation Using Mineralized vs. Demineralized Freeze Dried Bone Allograft. J Periodontol. 2011;

66. d’Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ. 2007 Jun 9;1162–71. doi: 10.1038/sj.cdd.4402121 17347663

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