Restor Dent Endod.  2014 Aug;39(3):187-194.

The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

• Jeong Y
• Yang W
• Ko H
• Kim M

Abstract

OBJECTIVES
The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells.
MATERIALS AND METHODS
MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer).
RESULTS
Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05).
CONCLUSIONS
These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

Keyword

Bone morphogenetic protein-2 (BMP-2); Enamel matrix derivative (EMD); MC3T3-E1 cell; Mineral trioxide aggregate (MTA)

MeSH Terms

Alkaline Phosphatase
Calcium
Dental Enamel*
Gene Expression
Integrin-Binding Sialoprotein
Osteoblasts
Osteocalcin
Osteonectin
Osteopontin
Polymerase Chain Reaction
Regeneration
RNA, Messenger
Pemetrexed
Alkaline Phosphatase
Calcium
Integrin-Binding Sialoprotein
Osteocalcin
Osteonectin
Osteopontin
RNA, Messenger

Figure

• 

  Figure 1 Alkaline phosphatase (ALP) expression in MC3T3-E1 cells in control and experimental groups. MC3T3-E1cells were cultured with or without treated materials for 7 days and stained with alkaline-dye mixture provided by an ALP staining kit. A representative photograph of ALP staining is shown, (a) control; (b) EMD; (c) BMP-2; (d) MTA; (e) MTA/EMD; (f) MTA/BMP-2. EMD, enamel matrix derivative; BMP, bone morphogenetic protein; MTA, mineral trioxide aggregate.

• 

  Figure 2 Formation of calcification nodules in each group in MC3T3-E1 cells. MC3T3-E1 cells were cultured with or without treated materials for 7 days and stained with Alizarin red. A representative photograph of Alizarin red staining is shown, (a) control; (b) EMD; (c) BMP-2; (d) MTA; (e) MTA/EMD; (f) MTA/BMP-2. EMD, enamel matrix derivative; BMP, bone morphogenetic protein; MTA, mineral trioxide aggregate.

• 

  Figure 3 The effects of BMP-2 and EMD on messenger RNA expression for ALP, BSP, OCN, OPN and OSN in MC3T3-E1 cells on days 3 (a) and 7 (b). The total mRNA was extracted from the cells, and mRNA expression was determined using RT-PCR. CON, control; BMP-2, bone morphogenetic protein-2; EMD, enamel matrix derivative; MTA, mineral trioxide aggregate; ALP, alkaline phosphatase; BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin; OSN, osteonectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse-transcription polymerase chain reaction analysis.

• 

  Figure 4 The fold change in the mRNA expression for ALP, BSP, OCN, OPN and OSN in MC3T3-E1 cells relative to the control on days 3 (a) and 7 (b). The control was assigned an optical density reference value of 1.0 (*p < 0.05). BMP-2, bone morphogenetic protein-2; EMD, enamel matrix derivative; MTA, mineral trioxide aggregate; ALP, alkaline phosphatase; BSP, bone sialoprotein; OCN, osteocalcin; OPN, osteopontin; OSN, osteonectin.

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