J Periodontal Implant Sci.  2017 Oct;47(5):273-291. 10.5051/jpis.2017.47.5.273.

Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells

• Kim EC
• Park J
• Kwon IK
• Lee SW
• Park SJ
• Ahn SJ

• KMID: 2392716
• DOI: http://doi.org/10.5051/jpis.2017.47.5.273

Abstract

PURPOSE
Although static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects.
METHODS
Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry.
RESULTS
The activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated.
CONCLUSIONS
SMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

Keyword

Bone regeneration; Periodontal guided tissue regeneration; Relative biological effectiveness; Signal transduction; Wnt proteins

MeSH Terms

Alkaline Phosphatase
Blotting, Western
Bone Regeneration
Calcium
Dental Cementum*
Dental Prosthesis
Gene Expression
Glycogen Synthase
Guided Tissue Regeneration, Periodontal
Humans
Immunohistochemistry
JNK Mitogen-Activated Protein Kinases
Magnetic Fields*
Miners
Osteoblasts*
Periodontal Diseases
Periodontal Ligament*
Phosphorylation
Polymerase Chain Reaction
Protein Kinases
Regeneration
Relative Biological Effectiveness
Reverse Transcription
RNA, Messenger
Signal Transduction
Wnt Proteins
Alkaline Phosphatase
Calcium
Glycogen Synthase
JNK Mitogen-Activated Protein Kinases
Protein Kinases
RNA, Messenger
Wnt Proteins

Figure

• 

  Figure 1 Effects of SMFs on osteoblastic differentiation in human osteoblasts. (A) Schematic diagram of the SMF exposure system. (B) Cell proliferation was examined by an MTT assay at 3, 7, and 14 days. (C) Differentiation was assessed based on ALP activity, (D) Alizarin red staining, (E) calcium content, and (F) expression of the mRNA of bone matrix proteins. The intensity of Alizarin red staining was determined by optical density. Cells were treated with OS medium containing 50 μg/mL of L-ascorbic acid and 10 mM β-glycerophosphate along with 3-, 15-, and 50-mT SMFs for 7 days and 14 days. OM contained OS and 10−7 M dexamethasone. The results are representative of 5 independent experiments that were performed. SMF: static magnetic field, MTT: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, ALP: alkaline phosphatase, OS: osteogenic supplement, OM: osteogenic medium, Runx2: runt-related transcription factor 2, OPN: osteopontin, OCN: osteocalcin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase. a)Significant difference compared to control (P<0.05); b)Significant difference between each group (P<0.05).

• 

  Figure 2 Effect of SMFs on osteoblastic differentiation in a cell line of human PDLCs and cementoblasts. (A, B) Cell proliferation was examined by an MTT assay at 3, 7, and 14 days. (C, D) ALP activity was determined and normalized to protein content. (E, F) Matrix mineralization was evaluated by Alizarin red staining. Cells were treated with 15-mT SMFs and OS or OM for 14 days. The results are representative of 5 independent experiments. SMF: static magnetic field, PDLC: periodontal ligament cell, MTT: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, ALP: alkaline phosphatase, OS: osteogenic supplement, OM: osteogenic medium. a)Significant difference compared to control (P<0.05); b)Significant difference between each group (P<0.05).

• 

  Figure 3 Effects of SMFs on calcium content and osteoblastic or cementoblastic gene expression in a cell line of human PDLCs and cementoblasts. (A, B) Ca2+ concentration was measured using a calcium quantification assay kit. (C, D) mRNA was determined by RT-PCR analysis. Cells were treated with a 15-mT SMF and OS or OM for 14 days. The results are representative of 5 independent experiments. SMF: static magnetic field, PDL: periodontal ligament, PDLC: periodontal ligament cell, RT-PCR: reverse transcription polymerase chain reaction, OS: osteogenic supplement, OM: osteogenic medium, Runx2: runt-related transcription factor 2, OPN: osteopontin, OCN: osteocalcin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, CEMP-1: cementum protein 1, CAP: cementum-derived attachment protein. a)Significant difference compared to control (P<0.05); b)Significant difference between each group (P<0.05).

• 

  Figure 4 Effect of SMFs on proliferation, ALP activity, and mineralized nodule formation in primary cultured human PDLCs and osteoblasts. (A, B) Cell proliferation was examined by an MTT assay at 3, 7, and 14 days. (C, D) ALP activity was determined and normalized to protein content. (E, F) Matrix mineralization was evaluated by Alizarin red staining as described in the Materials and Methods. Cells were treated with 15-mT SMFs and OS or OM for 14 days. The results are representative of 5 independent experiments. SMF: static magnetic field, ALP: alkaline phosphatase, PDLC: periodontal ligament cell, MTT: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, OS: osteogenic supplement, OM: osteogenic medium. a)Significant difference compared to control (P<0.05); b)Significant difference between each group (P<0.05).

• 

  Figure 5 Effect of SMFs on calcium content and osteoblastic gene expression in primary cultured human PDLCs and osteoblasts. (A, B) Ca2+ concentration was measured using a calcium quantification assay kit. (C, D) mRNA was determined by RT-PCR analysis. The results are representative of 5 independent experiments. SMF: static magnetic field, PDLC: periodontal ligament cell, RT-PCR: reverse transcription polymerase chain reaction, Runx2: runt-related transcription factor 2, OPN: osteopontin, OCN: osteocalcin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase. a)Significant difference compared to control (P<0.05); b)Significant difference between each group (P<0.05).

• 

  Figure 6 Effects of SMFs on the Wnt/β-catenin, MAPK, and NF-κB signaling pathways in human osteoblasts. (A, B) Cells were treated with SMFs and OS or OM for 2 days, (C) 30 minutes, and (D, E) 45 minutes. (A-D) Protein levels were assessed by western blot analysis and (E) immunofluorescence staining. Arrows (yellow) indicate the nuclear translocation of NF-κB p65. The data presented are representative of five independent experiments. SMF: static magnetic field, MAPK: mitogen-activated protein kinase, NF-κB: nuclear factor-κB, OS: osteogenic supplement, OM: osteogenic medium, GSK-3β: glycogen synthase kinase-3β, ERK: extracellular signal-regulated kinase, JNK: c-Jun N-terminal kinase.

• 

  Figure 7 Effects of various inhibitors of signal transduction on SMF-induced osteoblastic differentiation in human osteoblasts. (A-D) Cells were pretreated for 2 hours with DKK1 (0.5 μg/mL), SB203580 (20 μM), PD98059 (20 μM), SP600125 (10 μM), and PDTC (10 μM), and then cultured in OS with 15-mT SMFs for 14 days. The results are representative of 5 independent experiments. SMF: static magnetic field, OS: osteogenic supplement, Runx2: runt-related transcription factor 2, OPN: osteopontin, OCN: osteocalcin, GAPDH: glyceraldehyde 3-phosphate dehydrogenase. a)Significant difference between each group (P<0.05).

• 

  Figure 8 Schematic diagram illustrating the Wnt, Akt, MAPK, and NF-κB signaling pathways triggered by exposure to SMFs, which ultimately stimulate the osteoblastic differentiation of human osteoblasts. MAPK: mitogen-activated protein kinase, NF-κB: nuclear factor-κB, SMF: static magnetic field, Fzd: Frizzled, GSK-3β: glycogen synthase kinase-3β, JNK: c-Jun N-terminal kinase.

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