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Epigenetic Changes During Mechanically Induced Osteogenic Lineage Commitment

[+] Author and Article Information
Julia C. Chen

Department of Biomedical Engineering,
Columbia University,
New York, NY 10027

Mardonn Chua

Department of Biotechnology,
University of British Columbia,
Vancouver, BC V6T 1Z4, Canada

Raymond B. Bellon

Department of Chemical Engineering,
Columbia University,
New York, NY 10027

Christopher R. Jacobs

Mem. ASME
Department of Biomedical Engineering,
Columbia University,
New York, NY 10027

1Corresponding author.

Manuscript received December 13, 2014; final manuscript received January 4, 2015; published online January 26, 2015. Editor: Victor H. Barocas.

J Biomech Eng 137(2), 020902 (Feb 01, 2015) (6 pages) Paper No: BIO-14-1628; doi: 10.1115/1.4029551 History: Received December 13, 2014; Revised January 04, 2015; Online January 26, 2015

Osteogenic lineage commitment is often evaluated by analyzing gene expression. However, many genes are transiently expressed during differentiation. The availability of genes for expression is influenced by epigenetic state, which affects the heterochromatin structure. DNA methylation, a form of epigenetic regulation, is stable and heritable. Therefore, analyzing methylation status may be less temporally dependent and more informative for evaluating lineage commitment. Here we analyzed the effect of mechanical stimulation on osteogenic differentiation by applying fluid shear stress for 24 hr to osteocytes and then applying the osteocyte-conditioned medium (CM) to progenitor cells. We analyzed gene expression and changes in DNA methylation after 24 hr of exposure to the CM using quantitative real-time polymerase chain reaction and bisulfite sequencing. With fluid shear stress stimulation, methylation decreased for both adipogenic and osteogenic markers, which typically increases availability of genes for expression. After only 24 hr of exposure to CM, we also observed increases in expression of later osteogenic markers that are typically observed to increase after seven days or more with biochemical induction. However, we observed a decrease or no change in early osteogenic markers and decreases in adipogenic gene expression. Treatment of a demethylating agent produced an increase in all genes. The results indicate that fluid shear stress stimulation rapidly promotes the availability of genes for expression, but also specifically increases gene expression of later osteogenic markers.

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Grahic Jump Location
Fig. 2

Gene expression changes after treatment with CM from fluid shear stress stimulated osteocytes. mRNA levels for (a) Runx2, (b) Dlx5, (c) OSX, (d) OPN, (e) OCN, (f) PPARγ, (g) FABP4, and (h) LPL (bars indicate mean ± SE, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Grahic Jump Location
Fig. 1

Gene expression changes after treatment with demethylating agent. mRNA levels for (a) Runx2, (b) Dlx5, (c) OSX, (d) OPN, (e) OCN, (f) PPARγ, (g) FABP4, and (h) LPL (bars indicate mean ± SE, n = 4, **p < 0.01, ***p < 0.001).

Grahic Jump Location
Fig. 3

CpG methylation levels in cells treated with CM from static (open circles) or fluid shear stress stimulated (closed circles) osteocytes. Location of CpG site is shown as number of base pairs relative to transcription start site (0). Levels for (a) Runx2, (b) Dlx5, (c) OSX, (d) OPN, (e) OCN, (f) PPARγ, (g) FABP4, and (h) LPL (percentages were calculated from DNA from at least 10 colonies, n = 1).

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