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Fibrin Filled Scaffolds for Bone
Tissue Engineering: An in vivo Study
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Institute of Biomaterials and Biomedical Engineering
Jeffrey M. Karp, Feryal Sarraf, Molly S. Shoichet and John E. Davies
University of Toronto, CANADA
Background
th
6
International Meeting
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December 10-13 , 2003
Orlando, Florida
Methods and Results
- Fibrin Sealants (FS) used clinically in combination with autologous and
allogenic bone grafts (Tajima et al 1994, Sielewicz et al 1994), to
deliver antibiotics (Marchetti et al 1994) and hydroxyapatite particles
(Ferrari et al 1994).
- Effects of FS during bony wound healing have been inconsistent
(Schlag et al 1988).
- Properties of the fibrin relevant to host tissue invasion generally
ignored. FS investigated to enhance wound healing without
modification to suit application (Huang 2002 et al, Yamada et al 2003,
van Susante et al 1999, Bach et al 2001).
- Most FS are primarily designed to stop bleeding & provide adhesive
properties.
The main goal of this work was to determine how filling bone engineering
scaffolds with fibrin sealant effects bony wound healing and to examine whether
the structural properties of the fibrin are important to consider. Tisseel VH was
used as supplied by Baxter Bioscience Canada. To create fibrin matrices with
different structural properties, fibrinogen was combined with different
concentrations of thrombin. Biodegradable scaffolds filled with the different fibrin
matrices were implanted into the distal femur of adult inbred Brown Norway rats
and examined histomorphometrically after 2, 5 and 11 days. The results from the 5
day time point support many published in vitro cell migration studies where various
cells types were demonstrated to migrate more rapidly into fibrin matrices with
larger pores and thicker strands. This work demonstrates this phenomenon for the
first time in vivo. However, results from the 11 day time point showed that filling
scaffolds with commercially available fibrin sealants significantly delayed bony
wound healing. Therefore, fibrin sealants, in their present state, are likely not ideal
for bony wound healing applications.
- Fibrin gels with thin fibers/small pores significantly reduce cell
migration in vitro compared to fibrin gels with thick fibers/larger pores,
which may be requirements of a gel structure critical for normal wound
healing (Amrani et al 2001, Carr et al 1988).
- Most FS contain (only) a high concentration of thrombin, (Jackson
2001) which results in a network with thin short fibers with small pores
(Ryan et al 1999).
Figure 4. Histomorphometric analysis of reparative bone after 5
days in vivo. No statistical difference was found between empty
scaffolds and fibrin filled scaffolds containing fibrin(low T).
However, significantly less bone was found in defects containing
fibrin(high T) compared to empty scaffolds. These results correlate
with decrease in fibrin sealant amount between the 2 day and 5
day time points. (Low T ~ 62% reduction, high T ~ 34 %, p<0.06)
Hypotheses
1. Scaffolds filled with fibrin sealant will enhance bone tissue
invasion in comparison to empty scaffolds.
2. Fibrin matrices with large pores and thick strands will enhance
bone tissue invasion in vivo in comparison to matrices with
smaller pores and thinner fibrin strands.
Figure 2. Scanning electron micrographs of
5 Day
polymerized Tisseel fibrin sealant. (A) Fibrin
polymerized with a low thrombin concentration
(1.75 NIH U/ml) contained thick fibrin strands with
large pores compared to (B) fibrin polymerized with
a high thrombin concentration (220 NIH U/ml),
which contained thin fibrin strands with small
pores.
A
B
Figure 5. Histomorphometric analysis of reparative bone after
11 days in vivo. Both scaffolds filled with fibrin contained
significantly less bone then the empty scaffolds (p<0.003).
Figure 1. Scanning electron micrograph of
an empty PLGA-CaP
longitudinally.
scaffold
Conclusions
viewed
• Filling Scaffolds with fibrin significantly reduces blood cells from
becoming trapped within the pores of the scaffold.
C
5 Days
11 Days
Empty Scaffold
3
3
3
Fibrin Filled
Scaffolds (Low T)
4
4
3
Fibrin Filled
Scaffolds (High T)
4
4
Table 1. Number of defects per group
11 Day
2 Days
D
• During the early stages of bony wound healing, scaffolds filled with
fibrin polymerized with a low thrombin concentration supported more
bone invasion compared to scaffolds filled with fibrin polymerized with a
high thrombin concentration.
• Filling scaffolds with fibrin sealant delayed bony wound healing as
evidenced after 11 days in vivo. Therefore, fibrin sealants in their
present state are likely not ideal for bony wound healing applications.
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Figure 3.
F
(A) Bone was observed invading at the periphery of (low T) fibrin filled
scaffolds whereas (B) much less reparative bone was identified in (high T) fibrin filled
scaffolds. Large areas occupied by residual fibrin sealant (black arrow) were observed.
(C) Reparative bone at periphery of defects was bright red-orange (arrows), which
indicates presence of thick, mature collagen fibers. (D) Collagen fibers at the periphery
of defects were green (white arrows), which indicates thin and less mature. In
comparison to empty scaffolds, which were completely filled with bone(not shown)
(E,F), bone was only observed at the periphery of both fibrin filled scaffolds groups.
(A,B,E,F) Masson’s Trichrome stained sections. (C,D) Picro Sirus Red stained
sections.
Acknowledgements
Stuart Rae, Raisa Yakubovitch, Wanda Oprea, Dr. Tania Benatar, Dr.
David Lickorish & Professor John Weisel.
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.