Impact of Mechanobiology on Gene Expression in Human Osteoblasts

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Impact of Mechanobiology on Gene Expression in
Human Osteoblasts
Markin-Flanagan Undergraduate Student Research
Project for Bone and Joint Health

• Vitomir Tasevski, Jovina Sorbetti,
• and Sharon Chiu

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Introduction

• Bone cells in vivo are constantly exposed to
  physiological forces.
• The majority of cell studies are conducted in
  vitro without biomechanical stimuli.
• Cell cultures were exposed to hydrostatic
  pressure (HP) using a specially designed pressure
  chamber.
• MG-63 human osteosarcoma cell line was tested.

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Aims

• The specific aims of this study were to
• validate the pressure apparatus.
• characterise the MG-63 cell line.
• examine the effect of the hormone 1,25 dihydroxy
  vitamin D3 (1,25(OH)2D3).
• determine the effects of HP on the cells.
• compare the effects of static and cyclic HP.

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Methods Cells

• Cells were routinely cultured with DMEM-F12
  medium containing 10 fetal calf serum.
• Cells were seeded into 6 well plates at 2 x 105
  cells/well and grown for 48 hours.
• The medium was changed to serum free or serum
  free with 1,25(OH)2D3 and incubated for 20 hours.

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Methods Apparatus

• Cell culture plates were overlaid with sterile
  canola oil at 37oC.
• The chamber was filled with oil and immersed in a
  37oC water bath.
• 1 MPa of pressure was applied in either a static
  or a cyclic fashion.
• All tests were run for a total time of 4 hours.

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Canola Oil Overlay
Cell Growth Media and Cell Monolayer
Figure 1 Cross Section of 6-well Culture Plate
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Piston of MTS Steel Chamber Rack for Cell Culture
Plates Canola Oil Water Bath at 37oC
Figure 2 Cross Section of the Pressure Apparatus
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Methods Analysis

• Cells were washed with PBS and lysed with Trizol.
• RNA was successfully extracted and PCR was
  carried out with the cDNA.
• Validated primers for a series of genes were
  used
• GAPDH, Cbfa1, and osteocalcin.
• MMP-1, -2, -3 and 13.
• Collagen-I, biglycan, lumican, Timp-1, cfos, and
  cjun.

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Results

• Cells subjected to static or cyclic HP showed an
  increase in mRNA levels of MMP-1 and -3.
• Cells treated with 1,25(OH)2D3 showed
• an increase in osteocalcin expression.
• a decrease in expression of MMP-1 and -3.
• Cells exposed to 1,25(OH)2D3 and HP showed an
  increase in expression of MMP-1 and -3.
• No change in mRNA levels of any of the other
  genes was measured.

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• Figure 3 Effect of static HP (1.0MPa) on mRNA
• levels for GAPDH, MMP-1 and 3
• p lt 0.001 compared to controls

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Figure 4 Effect of cyclic HP (0-1.0MPa) on
mRNA levels for GAPDH, MMP-1 and -3 p lt 0.001
compared to controls
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Figure 5 Effect of cyclic HP (0-1.0MPa) on
Osteocalcin mRNA levels p lt 0.001 compared to
controls
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Figure 6 Effect of cyclic HP (0-1.0MPa) on MMP-1
and -3 mRNA levels p lt
0.001 compared to control p lt 0.01
compared to 1,25(OH)2D3 or to Pressure
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Discussion MMP-1 and -3

• Changes in MMP-1 and -3 suggests that
  biomechanical loading affects bone remodelling
• MMP-1 breaks down collagens I, II, III and X.
• MMP-3 breaks down collagen III and IV,
• laminin, fibronectin, and proteoglycan.
• The lack of change in the other genes indicates a
  specific cell response to HP.

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Discussion Osteoclacin

• Cbfa1 and Osteocalcin are markers of early and
  late osteoblast differentiation, respectively.
• They did not change with exposure to HP.
• Implies that neither static nor cyclic HP led to
  changes in differentiation of osteoblastic cells.

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Discussion Vitamin D3

• Increase in the mRNA levels of osteocalcin in
  cultures exposed to 1,25(OH)2D3 was associated
  with differentiation in cells.
• Cells were of a pre-osteoblastic phenotype.
• Cyclic HP had an effect on both pre- and
  differentiated osteoblasts.

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Conclusions

• Cells respond to their environment in a specific
  manner.
• Neither static nor cyclic HP led to changes in
  differentiation in cells.
• Cyclic HP affected pre- and differentiated
  osteoblasts.
• Biomechanical loading may play a significant role
  in bone remodelling.

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Future Directions

• A biomechanically active environment should be
  considered when studying normal or diseased bone
  homeostasis.
• This system can be used to examine the effects of
  HP on clonal or primary cells.
• The effects of hormones and drugs could be
  measured within a biomechanically active
  environment.