title: Calcium ions released from mineral trioxide aggregate convert the differentiation pathway of C2C12 cells into osteoblast lineage.
abstract: BACKGROUND
  The purpose of this study was to examine the effect of mineral trioxide aggregate (MTA) on pluripotent-mesenchymal cell differentiation.
  
  
  METHODS
  The pluripotent-mesenchymal cell line C2C12 was cultured in a 5% serum medium to induce cell differentiation with or without MTA. The differentiation to myoblasts was analyzed by the immunocytochemical staining of myosin heavy chains. The cellular phenotype-specific markers characterizing the osteoblasts (Runx2 and osterix), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (LPL) were estimated with mRNA and protein levels by using real-time polymerase chain reaction and Western blot analysis, respectively. To verify that the effect of MTA was caused by the released calcium ions, the mRNA levels were analyzed in the presence or absence of MTA with ethylene glycol tetraacetic acid, calcium chloride, or verapamil.
  
  
  RESULTS
  C2C12 cells cultured without MTA altered their phenotype to myoblasts, exhibiting positive reactions to myosin heavy chains. However, the cells cultured with MTA were strongly inhibited from developing into myoblasts. The mRNA and protein expressions of Runx2, osterix, and Sox9 significantly increased with MTA; the expressions of MyoD and LPL decreased significantly. Calcium chloride addition without MTA presented a significant increase of mRNA levels of Runx2, osterix, and Sox9; ethylene glycol tetraacetic acid addition with MTA presented a significant increase of mRNA levels of MyoD and LPL. Verapamil blocked the stimulating or suppressing effect of MTA on these transcription factors.
  
  
  CONCLUSIONS
  Our study showed that MTA converted the differentiation pathway of C2C12 cells into osteoblast and/or chondroblast lineages as a result of elution components such as calcium ions from MTA.
authors.affiliation: Nihon University Graduate School of Dentistry, Tokyo, Japan.
authors.forename: Sakurako
authors.initials: S
authors.lastname: Matsumoto
authors.forename: Makoto
authors.initials: M
authors.lastname: Hayashi
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Naoto
authors.initials: N
authors.lastname: Suzuki
authors.forename: Masao
authors.initials: M
authors.lastname: Maeno
authors.forename: Bunnai
authors.initials: B
authors.lastname: Ogiso
mesh_terms: D017667:Adipocytes / Q000187:drug effects; D017607:Aluminum Compounds / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D017610:Calcium Compounds / Q000737:chemistry*; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D019902:Chondrocytes / Q000187:drug effects; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D004338:Drug Combinations; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D008071:Lipoprotein Lipase / Q000032:analysis; D059630:Mesenchymal Stem Cells / Q000187:drug effects*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects; D017570:MyoD Protein / Q000032:analysis; D032446:Myoblasts / Q000187:drug effects; D018995:Myosin Heavy Chains / Q000032:analysis; D010006:Osteoblasts / Q000187:drug effects; D010087:Oxides / Q000737:chemistry*; D039904:Pluripotent Stem Cells / Q000187:drug effects*; D012387:Root Canal Filling Materials / Q000737:chemistry*; D055758:SOX9 Transcription Factor / Q000032:analysis; D017640:Silicates / Q000737:chemistry*; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000032:analysis; D014700:Verapamil / Q000494:pharmacology
chemical_list: D017607:Aluminum Compounds; D002121:Calcium Channel Blockers; D017610:Calcium Compounds; D002614:Chelating Agents; D050659:Core Binding Factor Alpha 1 Subunit; D004338:Drug Combinations; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; D010087:Oxides; C493709:RUNX2 protein, human; D012387:Root Canal Filling Materials; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D017640:Silicates; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; C086631:mineral trioxide aggregate; D004533:Egtazic Acid; D014700:Verapamil; C548261:LPL protein, human; D008071:Lipoprotein Lipase; D018995:Myosin Heavy Chains; D002122:Calcium Chloride; D002118:Calcium