T DEX

1
Examination of the Effects of Thrombin

on the Migration of Osteogenic Cells
484

• Introduction
• Thrombin (Factor IIa) is a 36.5 kD serine
  proteinase that is generated in the penultimate
  step of the blood coagulation cascade from the
  circulating plasma protein prothrombin (Factor
  II). Once formed, thrombin initiates fibrin
  polymerization by cleaving fibrinogen.
• Thrombin has been demonstrated to stimulate
  migration of a variety of cell types (Bar-Shavit
  et al. 1983, Dawes et al. 1993, Wang et al.
  1997), however no studies exist that examine
  thrombin's role on bone marrow derived osteogenic
  cell migration. Delivering thrombin or thrombin
  peptides from biodegradable scaffolds may help
  enhance bone regeneration within such scaffolds
  by promoting osteogenic cell invasion to, and
  throughout the defect site. In this study, a 2-D
  cell migration scratch wound model was used to
  examine the effects of thrombin on rat bone
  marrow derived adherent cells under osteogenic
  culture conditions. An osteogenic cell line was
  used to provide evidence that the observed
  effects applied to the osteogenic population of
  the primary cells.
• Hypothesis
• Bone marrow derived osteogenic cells will be
  stimulated to migrate by the addition of thrombin
  and this effect will be mediated by a direct
  interaction with thrombins active site.
• Objectives
• To study the effect of thrombin on bone marrow
  derived osteogenic cells to determine
• a. If thrombin stimulates chemokinesis
• If the migration response is mediated via
  thrombins active site

Thrombin Affects Cell Invasion
Fibrin Structural Properties
Receptor Ligand Interactions
Focus of this work
Low Thrombin
High Thrombin
Cell Migration
Figure 4. Samples of media (containing thrombin)
were examined over 24 hours from different
culture conditions with S-2238. Thrombin activity
decreased quickly in the presence of cells. Media
from cell line cultures without thrombin did not
interact with the thrombin substrate.
Results
Figure 5. After 10 hours of adherence to the
filter, cells on the upper surface were removed
and either 1BSA or 1BSA containing thrombin was
added for 24 hours. (A) More bone nodules were
observed on the underside of the filters when a
pulse of thrombin was added. However, (B) no
statistically significant difference was found
between the number of cells observed on the
bottom of filters treated with (T/T) and without
(C/C) thrombin.
A
Plt 0.04
Primary Bone Marrow Derived Cells Cultured in
Osteogenic Conditions
Rat Bone Marrow Derived Osteogenic Cell Line
(RSB4D-C8 )
Bone Nodules
of Control Leading Front of Migration

• T DEX

T DEX

• T -DEX

T -DEX
n3-7
Figure 1. Thrombin stimulated advancement of
both (left) the primary cells and (right) the
osteogenic cell line. A concentration of 1
U/ml was chosen for subsequent experiments.
of Cells that Migrated Through
the Filter
n6
Scratch Wound Model
Conclusions - Thrombin stimulates the migration
of both primary bone marrow derived cells in
osteogenic culture conditions and of an
osteogenic cell line. - This provides
evidence that thrombin may be interacting with
the osteogenic population of the primary
cells. - The migration response is mediated via
thrombins active site. - The activity of the
thrombin in culture quickly decreases, which is
likely cell mediated. - The ability of
thrombin (under Dex conditions) to increase the
capacity for bone formation at a distant site
does not correlate with an increased number of
migrating cells. This suggests that thrombin
may specifically interact with osteoprogenitor
cells, although more work is required to
confirm this. Future Work - Examine of cells
expressing the thrombin PAR-1 receptor. - Invest
igate if thrombin can also stimulate chemotaxis
of bone marrow derived osteogenic
cells. - Investigate the mechanism that mediates
differences in the modified Boyden chamber/bone
nodule assay. Acknowledgements
An average leading front of migration is
determined
Cells are grown to confluency
Cell layer is scratched with a pipette tip
Cell layer subjected to different conditions
Stuart Rae, Raisa Yakubovitch Professor Jaro
Sodek. This work was financially supported by an
ORDCF awarded to JED and by a University of
Toronto Doctoral Award and an OGS awarded to JMK.
Figure 3. When Mitomycin C was used to inhibit
cell proliferation and isolate the migration
response, results were consistent with those
displayed in Fig 2 (results for the primary cells
are shown).