Biomedical Engineering Reference
In-Depth Information
signaling by growth factors present in the ECM
of bone.
The ECM proteins interact with integrins, a
heterodimeric cell-membrane receptor family.
The
mineralization was observed in the uncoated
titanium meshes and meshes coated with fi bro-
nectin when antibodies against fi bronectin or
type I collagen integrins were present. Meshes
coated with both type I collagen and fi bronec-
tin showed signifi cantly higher calcium content,
a marker for matrix mineralization, when cul-
tured in the presence of antibodies against col-
lagen or fi bronectin integrins. A similar
phenomenon also was observed for collagen-
coated meshes cultured in the presence of anti-
bodies against fi bronectin integrins. No
signifi cant differences in osteocalcin content
were observed among the treatment groups.
However, all groups exposed to antibodies
against fi bronectin integrins showed a signifi -
cant decrease in osteocalcin content. Therefore,
fi bronectin or type I collagen coating does not
signifi cantly stimulate the differentiation of rat
bone marrow cells seeded in titanium fi ber
mesh. On the other hand, interactions between
fi bronectin and type I collagen integrins and
the substratum seem to be important during
the proliferation and early osteoblastic differ-
entiation of rat marrow stromal osteoblasts on
titanium surfaces.
α
5
β
1
integrins appear to be the
integrins found on osteoblasts that interact
specifi cally with fi bronectin, whereas
α
4
β
1
and
α
1
β
1
and
α
2
β
1
integrins recognize type I collagen [
23
,
25
,
30
]. The integrin-mediated contact of osteo-
blasts with fi bronectin or type I collagen
increases the expression of growth factors of
the TGF-
/BMP family, which in turn stimu-
lates osteoblast differentiation. The induction
of osteoblastic differentiation requires interac-
tion of the fi bronectin coating or type I colla-
gen coating with integrin receptors on the cells.
Integrins mediate the transmission of informa-
tion from the ECM by serving as a direct link
between the ECM and the intracellular actin
cytoskeleton. Signal transduction involves
tyrosine phosphorylation, which is linked to
the mitogen-activated protein kinase (MAPK)
pathway and other pathways [
β
]. Focal adhe-
sion kinase and MAPK are involved in the
induction of alkaline phosphatase activity in
osteoblasts.
Van den Dolder et al. [
31
] investigated the
effect of fi bronectin or a type I collagen coating
on the differentiation of primary osteoblasts in
titanium fi ber mesh scaffolds. Rat bone marrow
cells were cultured for several days in plain and
coated titanium fi ber meshes in the presence of
antibodies against fi bronectin and type I col-
lagen integrins. The results showed no signi-
fi cant effects of the coatings on cellular
proliferation, as indicated by DNA quantifi ca-
tion. When antibodies against fi bronectin and
type I collagen integrins were used, a signifi -
cant reduction in cell proliferation was observed
for the uncoated titanium meshes, meshes
coated with collagen, and meshes coated with
collagen and fi bronectin. The different coat-
ings also did not affect the alkaline phospha-
tase activity, an early marker for differentiation,
of the cells seeded on the coated and uncoated
meshes. However, the presence of antibodies
against fi bronectin or type I collagen integrins
signifi cantly delayed the expression of alkaline
phosphatase activity for uncoated titanium
meshes, meshes coated with collagen, and
meshes coated with collagen and fi bronectin.
No signifi cant effect of fi bronectin or type I
collagen coating on matrix mineralization was
obser ved . Fu r t hermore, no d i f ference i n mat r i x
35
5.5 In Vivo Bone
Engineering: Cell-Based
Approach
Previous studies with cell-loaded titanium fi ber
mesh showed that culture time is important for
the fi nal in vivo bone formation. Vehof et al.
[
] found that cell-loaded titanium fi ber mesh,
without prolonged culture, produced a very
limited amount of bone after subcutaneous
implantation in rats. On the other hand, pro-
longed culture (
41
days) of cell-loaded titanium
fi ber mesh resulted in abundant mineralization
without a bonelike tissue organization [
8
]. To
resolve the contradiction between short and
long culture, we independently evaluated the
effect of culture time on bone formation by rat
bone marrow cells seeded in titanium fi ber
mesh. Osteogenic cells were cultured for
45
1
,
4
,
and
days on titanium fi ber mesh and then
implanted subcutaneously in rats [
8
]. Analysis
of DNA as an index of cell grow th in the in vitro
experiment revealed a lag phase from days
39
1
through
4
, but a
42
% increase in DNA content
