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(fetuin) inhibits pathologic calcification
in vitro
and
in
vivo
77
and may be elevated in OI in an attempt to pre-
vent calcification outside the collagen matrix. Bone sia-
loprotein is an
in vitro
promoter of calcification78,79
78,79
and
regulates bone formation
in vitro
and in some animal
models.
78,80
It is of interest to note that the fro
/
fro mouse
model of OI had reduced levels of osteonectin, but also
reduced levels of bone sialoprotein, which is different
than the human situation.
81
The effects of these noncol-
lagenous proteins are most likely determined where the
proteins bind relative to the mutation, and whether they
can still associate with the collagen in their appropriate
orientation or if their binding is prevented or their orien-
tation is altered. Thus, some or all of these proteins may
be playing a role in determining the mineralization pro-
cess in these individuals.
Osteoblast cell cultures derived from human OI bone
biopsies were screened for production of NCPs and accu-
mulation of these proteins in the extracellular matrix of
the cultures.
82
In the OI cultures there was decreased
formation of osteonectin and three proteoglycans (a
large chondroitin sulfate proteoglycan, biglycan and
decorin). Additionally, increased steady-state levels of
fibronectin and thrombospondin synthesis and incor-
poration into the culture extracellular matrix relative to
the age-matched controls was found. Hyaluronan was
also increased in the matrix of these cultures relative to
cultures of control cells without changes in the steady-
state levels. The large and small proteoglycans bind to
collagen and other matrix substituents
83
but they have
also been shown to regulate the mineralization pro-
cess.
46
Fibronectin binds to collagen and other matrix
proteins and can affect osteoblast differentiation.
84
The
thrombospondins have a variety of different effects on
mineralization and at the time the culture experiments
mentioned were performed it was not known that there
were many different types of thrombospondins. For
example, thrombospondin 2 stimulates mineralization;
thrombospondin 1 blocks osteoblast differentiation.
85,86
In general, the alterations in protein expression in cul-
ture may only reflect the activity of the osteoblasts or may
indicate altered synthesis of specific proteins that cannot
bind to their “binding sites”
87
on the type I collagen triple
helix. Altered binding to these sites could also affect min-
eralization mechanisms, either because nucleation sites
on the collagen or matrix proteins are exposed enhancing
mineralization or because they are blocked (decreasing
mineralization). More recent proteomic and genetic stud-
ies are just beginning to address these mechanisms.
More modern techniques such as proteomics and gene
discovery have been performed to a lesser extent on OI
bones or cells derived from OI bones. The molecular fac-
tors contributing to the brittle phenotype in the trans-
genic BrtlIV mice included increased RANK, RANKL
and osteoprotegerin (OPG) levels based on RT-PCR.
88
More detailed studies of these mice with real-time qRT-
PCR analysis revealed significantly increased expression
of transcripts of late osteoblastic and osteocyte markers
vs. wild-type femurs.
89
In the first two-dimensional dif-
ferential display analyses of OI calvarial osteoblasts from
lethal and viable Brtl mice the lethal mice had increased
expression of an endoplasmic reticulum stress-related
protein, whereas expression of the chaperone alphaB
crystallin was increased in nonlethal mice. Lethal BrtlIV
mice had increased expression of several cartilaginous
proteins and lower expression of matrilin 4, microfibril-
associated glycoprotein 2 and thrombospondin-3.
90
Thrombospondin-3 acts like thrombospondin-1 in stim-
ulating new bone formation.
91
Although limited to date
such studies provide some credence to the concept that
defects in collagen can influence the binding or expres-
sion of other extracellular matrix proteins, which in turn
influences the ability of the defective collagen matrix to
be mineralized.
NORMAL AND OI MINERALIZATION
PROCESSES
The combination of the above information and the
variation in OI phenotype associated with different
mutations provide some insight, although not conclu-
sive, into the mineralization process in OI, and from the
lessons learned, into normal bone formation. There are
two distinct processes of bone formation during devel-
opment, endochondral ossification in which calcified
cartilage forms and is replaced by bone, and direct min-
eral apposition onto type I collagen (intramembranous
bone formation). Treatment for OI often takes advantage
of the endochondral pathway by stabilizing the calcified
cartilage and giving newly formed bones more strength
(remember that cartilage collagen, type II, is not affected
in OI). A schematic of the normal osteoblast-mediated
mineralization process and where there might be defects
in different forms of OI is seen in
Figure 4.7
. The first step
(not shown) is the formation and recruitment of osteo-
blasts (on the surface of bone or newly formed calcified
cartilage (primary mineralization)) followed by the depo-
sition of a mineralizable matrix by the osteoblasts. This
matrix consists of collagen and noncollagenous proteins
and enzymes, cytokines and lipids; the functions of each
have been reviewed elsewhere.
46
Since in OI, regardless
of type, the osteoblast is producing something abnormal
(mainly defective collagen) it is not surprising that defects
in other proteins might be found. Some of these, as dis-
cussed above, have already been identified. It is not clear
whether the altered chemical composition just reflects the
destruction of cells that are making an improper collagen
and the other constituents are destroyed at the same time
or whether there are altered levels of expression.
