analyzed by confocal microscopy. Transmission electron microscopy (TEM) was used to verify the presence of de novo bone formation within the fibrin gels.

1
Platelet Releasate Stimulates Bone Marrow Cell
Migration and Proliferation In Vitro W.E.
Oprea, J.M. Karp, M.M. Hosseini, J.E.
Davies Institute of Biomaterials and Biomedical
Engineering, University of Toronto, Toronto CANADA
analyzed by confocal microscopy. Transmission
electron microscopy (TEM) was used to verify the
presence of de novo bone formation within the
fibrin gels. Proliferation Cells plated at a
density of 104 cells/cm2 were subjected to the
effects of PR or rhPDGF-BB at parallel time
points to the 3-D migration studies. For
counting, cells were trypsinized and counted
using a hemocytometer. Results Confocal
microscopy revealed extensive bone marrow cell
infiltration into the fibrin matrix (Figure 1).
Determination of the leading front of migration
showed migration ranging from 50 to 350?m
(equivalent to 70 ?m/day), which corresponds well
with published in vivo data 4. TEM analysis
(Figure 2) showed the presence of electron dense
bodies containing hydroxyapatite within the
fibrin gels, thus confirming that a percentage of
the migratory cells were indeed osteogenic.
Figure 1. Series of horizontal
images obtained by confocal microscopy, depicting
cell infiltration into the fibrin gel. Image
series begins at bottom of the culture dish (a)
where a confluent cell layer is visible, and
proceeds up through the unstained fibrin gel.
Cells are stained green for F-actin, rending the
cell body and extended cell processes clearly
visible. Vertical distance between images 15?m.
Field width 265 ?m.   Figure 2. (A)
TEM photomicrograph depicting the branch of an
osteogenic cell (oC) embedded within the
extracellular fibrinous matrix (efM). In
proximity to the cell branch, several spherical,
electron dense bodies on the order of 0.5 ?m are
visible (arrows), which at higher magnification
(B) are shown to contain individual needle-shaped
particles (arrows). (C) The presence of
hydroxyapatite these foci of mineralization (FM)
was confirmed by the circular electron
diffraction pattern 5. Field width A 10 ?m.
Field width B 1.7 ?m.
PR was shown to stimulate the migration of RBMC
within the 3-D fibrin matrices. In groups with
added PR, a 25 increase in the leading front of
migration and a 3.5x increase in the number of
migrating cells (Figure 3) was observed. Similar
trends were observed when rhPDGF-BB was added
(not shown) however, the rhPDGF-BB groups were
not statistically significant from the control
groups. Figure 3. (A) Average
leading front of migration, or furthest distance
migrated by any one cell at analyzed point in ?m.
(B) Number of migrating cells in the volume above
a 250 x 250 ?m area. Experimental groups are
divided according to day(s) after subculture on
which PR was added. Proliferation studies
(Figure 4) further demonstrated that PR is
mitogenic for RMBC. Comparison between migration
and proliferation data indicated that PR also
stimulates the recruitment of RBMC to
migration. Figure 4 RBMC
proliferation. Growth factors were added at
parallel time points to the 3-D migration
studies. Conclusions Platelet releasate
stimulates the recruitment, migration and
proliferation of bone marrow derived cells. Our
results provide a potential mechanism to explain
why biologically active platelet-derived factors
may enhance endosseous wound healing.
Introduction Before placement of an implant at
an endosseous site, extravasated blood will fill
the wound compartment and begin to clot.
Concomitantly, platelets become activated and
release a library of growth factors and cytokines
within the injury site. It has been suggested
that these factors play an important role in the
regulation of the wound healing cascade by
stimulating the migration and proliferation of
osteogenic cells within the fibrin clot of the
injury compartment. Such thinking has encouraged
new clinical strategies that incorporate various
growth factors with materials in order to
accelerate the wound healing process 1.
However, for such strategies to be successful,
the underlying mechanisms of platelet growth
factor action at endosseous sites must be more
completely understood. To date, no information
on the effects of platelet factors on bone cell
migration is available 2,3. The purpose of this
work was to develop an in vitro model to study
the effects of the growth factors contained in
platelet releasate (PR) on osteogenic cell
migration and proliferation. Materials and
Methods Cell culture Rat bone marrow cells
(RBMC) were harvested from the femurs of young
Wistar rats and cultured in fully supplemented
media (FSM) consisting of ?-minimal essential
media supplemented with 15(v/v) fetal calf
serum, 10 (v/v) antibiotics, 50 ?g/ml L-ascorbic
acid, 5mM ?-glycerophosphate and 10-8 M
dexamethazone. Cultures were incubated at
37C and 100 relative humidity. Platelet
concentrate (PC) was obtained as prescribed by
the 3i PCCS system. To obtain PR, PC was
activated with 1 IU human thrombin/ml and the
supernatant containing the released platelet
factors was collected after centrifugation at
2000xg. 3-D Migration Confluent RMBC cultures
in 35 mm tissue culture dishes were overlaid with
a 1.5 mm thick fibrin gel, obtained by mixing 3
mg/ml human fibrinogen with 1.5 IU/ml thrombin.
These groups were supplemented with 2 ml FSM
containing PR (14 v/v) or rhPDGF-BB (20 ng/ml)
at various time points. On day 5 after gel
overlay, cultures were fixed and stained
with phalloidin, an F-actin stain, and

Acknowledgements The authors gratefully
acknowledge the provision of fibrin from
Haemacure Corp., and financial support from both
Implant Innovations Inc. and an Ontario Research
and Development Challenge Fund (ORDCF) grant to
JED.
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