J Periodontal Implant Sci.  2012 Jun;42(3):73-80. 10.5051/jpis.2012.42.3.73.

Effects of immunosuppressants, FK506 and cyclosporin A, on the osteogenic differentiation of rat mesenchymal stem cells

• Byun YK
• Kim KH
• Kim SH
• Kim YS
• Koo KT
• Kim TI
• Seol YJ
• Ku Y
• Rhyu IC
• Lee YM

• KMID: 2094730
• DOI: http://doi.org/10.5051/jpis.2012.42.3.73

Abstract

PURPOSE
The purpose of this study was to investigate the effects of the immunosuppressants FK506 and cyclosporin A (CsA) on the osteogenic differentiation of rat mesenchymal stem cells (MSCs).
METHODS
The effect of FK506 and CsA on rat MSCs was assessed in vitro. The MTT assay was used to determine the deleterious effect of immunosuppressants on stem cell proliferation at 1, 3, and 7 days. Alkaline phosphatase (ALP) activity was analyzed on days 3, 7, and 14. Alizarin red S staining was done on day 21 to check mineralization nodule formation. Real-time polymerase chain reaction (RT-PCR) was also performed to detect the expressions of bone tissue-specific genes on days 1 and 7.
RESULTS
Cell proliferation was promoted more in the FK506 groups than the control or CsA groups on days 3 and 7. The FK506 groups showed increased ALP activity compared to the other groups during the experimental period. The ALP activity of the CsA groups did not differ from the control group in any of the assessments. Mineralization nodule formation was most prominent in the FK506 groups at 21 days. RT-PCR results of the FK506 groups showed that several bone-related genes-osteopontin, osteonectin, and type I collagen (Col-I)-were expressed more than the control in the beginning, but the intensity of expression decreased over time. Runx2 and Dlx5 gene expression were up-regulated on day 7. The effects of 50 nM CsA on osteonectin and Col-I were similar to those of the FK506 groups, but in the 500 nM CsA group, most of the genes were less expressed compared to the control.
CONCLUSIONS
These results suggest that FK506 enhances the osteoblastic differentiation of rat MSCs. Therefore, FK506 might have a beneficial effect on bone regeneration when immunosuppressants are needed in xenogenic or allogenic stem cell transplantation to treat bone defects.

Keyword

Cell differentiation; Cyclosporin A; FK506; Immunosuppressive agents; Mesenchymal stem cells

MeSH Terms

Alkaline Phosphatase
Animals
Anthraquinones
Bone Regeneration
Cell Differentiation
Cell Proliferation
Collagen Type I
Cyclosporine
Durapatite
Gene Expression
Immunosuppressive Agents
Mesenchymal Stromal Cells
Osteoblasts
Osteonectin
Rats
Real-Time Polymerase Chain Reaction
Stem Cell Transplantation
Stem Cells
Tacrolimus
Alkaline Phosphatase
Anthraquinones
Collagen Type I
Cyclosporine
Durapatite
Immunosuppressive Agents
Osteonectin
Tacrolimus

Figure

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  Figure 1 Effects of FK506 and cyclosporin A (CsA) on the proliferation of rat mesenchymal stem cells. Cell viability was assessed with the MTT assay and the results were presented as optical density (OD) at 540 nm. All groups showed increased cell proliferation over time. The greater OD value of the FK506 groups indicated increased cell proliferation compared to the control and CsA groups on days 3 and 7. Significant difference from the control (a)) and C500 (b)) (P<0.05). F50: 50 nM FK 506 group, F500: 500 nM FK506 group, C50: 50 nM CsA group, C500: 500 nM CsA group.

• 

  Figure 2 Effects of FK506 and cyclosporin A (CsA) on the alkaline phosphatase (ALP) activity of rat mesenchymal stem cells. The FK506 groups showed higher ALP activity than other groups. There were no significant differences between the control and CsA groups. Significant difference from the controla), C50b), and C500c) (P<0.05).

• 

  Figure 3 Alizarin red S staining of rat mesenchymal stem cells after 21 days of subculture. Mineralization nodule formations were most prominent in FK506 groups. (A) Control, (B) 50 nM FK506, (C) 500 nM FK506, (D) 50 nM CsA, and (E) 500 nM CsA.

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  Figure 4 Osteopontin (A), osteonectin (B), Runx2 (C), Col-I (D) and Dlx5 (E) mRNA expression during cell differentiation of rat mesenchymal stem cells. Relative mRNA levels were plotted as the fold change of the bone-related gene over the control and normalized to the GAPDH level.

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