Full text of DOI:10.1359/jbmr.2001.16.3.497

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 3, 2001
©
2001 American Society for Bone and Mineral Research
The Bone Morphogenetic Proteins Antagonist Noggin
Inhibits Membranous Ossification
1
1
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PER ASPENBERG, CHARLOTTE JEPPSSON, and ARIS N. ECONOMIDES
ABSTRACT
Bone morphogenetic proteins (BMPs) are expressed and secreted during fracture repair. Although they are
likely to be required for this process, little is known about their physiological role in bone regeneration. Noggin
is a protein that specifically binds and inactivates several BMPs. It plays fundamental roles during early
embryonal development and limb morphogenesis by this BMP-inactivating activity. This study shows that
Noggin can modify bone formation in vivo in the adult animal and, thus, indirectly, that BMP signaling is
indispensable in this process. A noggin mutein (hNg⌬B2-Fc) engineered so as to display increased bioavail-
ability was used. Bilateral titanium bone chambers were inserted in 70 rats, and side comparisons for bone
formation in the chambers were done. The hNg⌬B2-Fc had no effect on total amount of tissue formed in the
chamber but decreased the amount of bone compared with both buffer controls and a control made up of an
Fc-tagged IL-6R␣ protein, which had no effects of its own. Also, wild-type noggin inhibited bone formation.
Thus, endogenous BMP signaling is necessary for normal bone regeneration. (J Bone Miner Res 2001;16:
4
97–500)
Key words: bone morphogenetic proteins, noggin, bone regeneration, bone chamber, rat
(
2)
INTRODUCTION
noggin/BMP pairs. For example, the complex of noggin/
BMP-2 displays an apparent K of approximately 20 pM,
d
URING RECENT years it has been discovered that the activity indicating a high-affinity interaction with a very low disso-
of signaling molecules belonging to the transforming ciation rate, and in concordance with this data, noggin has
D
growth factor ␤/bone morphogenetic protein (TGF-␤/BMP) been shown to be a very effective inhibitor of BMP-2 or
superfamily is modulated by a number of other secreted pro- BMP-4–induced signaling in biological assays. Noggin also
teins that function as specific antagonists at least during em- binds to BMP-7 (with lesser affinity than that for BMP-2
bryonic development. The best characterized of the BMP an- and BMP-4), and to BMP-5, BMP-6 (A. N. Economides, X.
tagonists is noggin, which was cloned in a screen of genes that Wang, N. Stahl, unpublished results, 1998), growth and
(3)
(4)
could rescue dorsal development in ventralized frog (Xenopus differentiation factor 5 (GDF-5), and GDF-6, but to
(1)
laevis) embryos. It was later shown that noggin exerts its activin, GDF-12, TGF-␤, and nodal (BMP-16; A. N. Econo-
biological effects by binding to BMPs and blocking their mides, X. Wang, N. Stahl, unpublished results, 1998). Dur-
(2)
binding to the BMP receptors. The name noggin comes from ing development, noggin often is expressed in conjunction
the observation that frog embryos with overexpression of this with BMPs, conceivably defining boundaries of BMP-
substance develop bigger heads.
induced structures. Noggin knockout mice die after birth.
Noggin displays selectivity for different BMPs both with They have coarse, thickened long bones, indicating that
respect to the BMPs it binds to and with respect to the noggin plays an inhibitory role in regulating periosteal
(
5)
affinity of the noggin/BMP complex for any of the possible bone growth.
1
Department of Orthopedics, Lund University Hospital, Lund, Sweden.
Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA.
2
4
97

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ASPENBERG ET AL.
FIG. 1. The bone chamber inserted in rat tibia. Arrows point at
ingrowth openings.
In this study, we asked the question whether noggin
would inhibit bone regeneration in an adult animal. If nog-
gin could be shown to inhibit, for example, fracture repair,
this would be clear evidence that BMP signaling plays an
important role during fracture repair. Although BMPs are
known to be expressed during bone regeneration, it has not
been shown clearly that they are necessary for this process.
FIG. 2. Specimens from bilateral chambers 4 weeks after implantation
(HE; specimen height, 7 mm). (A) Collagen carrier with buffer solution.
The bone ingrowth frontier has reached about half the way from the bottom
to the top of the chamber. Above that is a layer of fibrous or undifferen-
tiated tissue. Remaining carrier is seen on top, delineated from the fibrous
tissue by a thin dark line of inflammatory cells and bleeding. (B) Noggin
MATERIALS AND METHODS
(
hNg⌬B2-Fc), 10 ␮g. The fibrous tissue has reached halfway into the
We used a bone chamber technique, which is a model for
chamber, but there is only little bone at the bottom.
(6)
purely membranous ossification. The bone chamber con-
sists of a titanium screw with a cylindric interior space. It is
made up of two threaded half cylinders held together by a
hexagonal closed screw cap. One end of the implant is
Wild-type noggin was expressed in Escherichia coli and
screwed into the bone. The interior of the chamber has a refolded, and hNg⌬B2-Fc and IL-6R␣-Fc were produced in
diameter of 2 mm and is 7 mm long. There are two ingrowth Chinese hamster ovary (CHO) cells and purified to over
openings located at the bone end of the chamber (Fig. 1). 99% purity. These substances were kindly provided by
Thus, the ingrowing tissues enter the cylindrical space from Regeneron Pharmaceuticals, Inc. (Tarrytown, NY, USA).
the bone compartment. The chamber space extends far out
The various solutions were pipetted onto pieces of type I
into the subcutaneous region and the ingrown bone–derived collagen (Helistat; Colla-Tec, Inc., Plainsboro, NJ, USA)
tissue has the possibility of filling the chamber without sized to fill out the chamber in its dry state. The collagen
competition with other tissues. Ingrowing tissue, most of it was then freeze-dried before implantation. Different batches
bone, will fill a portion of the chamber within 4 weeks but of this collagen material appeared to differ in density and
not will reach the far end of the cylinder.
A genetically engineered noggin mutein lacking the heparin- batch was used for both sides in each experiment.
binding site and expressed as a chimeric protein with the Fc The chambers were inserted bilaterally in rats so that the
adsorption characteristics, but the collagen from the same
portion of human immunoglobulin G1 (IgG1; hNg⌬B2-Fc) evaluation could be based on side comparisons. Six side
was used, because it has been shown to display a better comparisons were done (Table 1): hNg⌬B2-Fc (1 ␮g) ver-
pharmacokinetic profile compared with wild-type noggin on sus vehicle control, hNg⌬B2-Fc (10 ␮g) versus vehicle
systemic administration in mice (A.N. Economides, X. Liu, control, hNg⌬B2-Fc (10 ␮g) versus IL-6R-Fc (10 ␮g),
F.H. Gannon, E.M. Shore, X. Wang, G. Elove, K.B. Shriram, IL-6R-Fc (10 ␮g) versus vehicle control, and wild-type
E.K. Fytros-Sirabian, J.P. Fandl, T.E. Ryan, K.M. Bailey, F.S. noggin (2 ␮g) versus vehicle control. The rats were killed
Kaplan, R.B. Kimble, N. Stahl, unpublished data, 2000). As a after 4 weeks, but the hNg⌬B2-Fc (10 ␮g) versus IL-6R-Fc
control, we used interleukin-6 receptor ␣ (IL-6R␣), also ex- (10 ␮g) experiment was done also with an observation time
pressed as a chimeric protein with the Fc domain of human of 7 weeks. All groups consisted of 10 rats each.
IgG1 fused in frame to its C terminus (IL-6R␣-Fc). IL-6R␣-Fc
Three hours before the rats of the 10-␮g hNg⌬B2-Fc
binds and blocks the activity of human IL-6 but does not affect versus vehicle group were killed, they were given a tech-
the activity of rat IL-6 and thus should be expected to play no netium labeled bisphosphonate, and immediately after har-
role in bone formation.
vest the radioactivity of the entire specimens was measured.

NOGGIN INHIBITS OSSIFICATION
499
TABLE 1. EFFECT OF NOGGIN IN PAIRED EXPERIMENTS (EXPERIMENT AND CONTROL SIDE IN THE SAME RAT; MEAN SD)
Experiment
Ng 1 ␮g
Ng 10 ␮g
Ng 10 ␮g
Ng 10 ␮g
IL-6R
Control
n
Tot c
Tot (e-c)
Bone c
Bone (e-c)
p (bone)
Vehicle
Vehicle
Vehicle
IL-6R
Vehicle
Vehicle
IL-6R
10
9
10
9
8
10
9
1.8 0.3
1.9 0.6
2.3 0.5
1.6 0.3
1.0 0.3
4.6 0.7
2.2 1.2
0.16 0.63
Ϫ0.30 0.88
Ϫ0.04 0.52
Ϫ0.19 0.44
0.06 0.28
0.03 0.87
0.14 1.1
1.0 0.2
0.7 0.6
1.5 0.6
0.8 0.3
0.6 0.3
1.3 0.46
1.6 1.0
Ϫ0.17 0.20
Ϫ0.24 0.64
Ϫ0.45 0.56
Ϫ0.32 0.33
0.06 0.39
0.03
0.29
0.03
0.02
0.66
0.03
0.97
a
wNg 2 ␮g
Ng 10 ␮g (7 weeks)
Ϫ0.37 0.44
0.01 0.88
Tot c, total tissue ingrowth distance in control; Tot (e-c), total tissue ingrowth distance in experiment side minus control; bone c, bone
ingrowth distance in control; bone (e-c), bone ingrowth distance in experiment side minus control; p (bone), p value for bone difference;
Ng, modified noggin (hNg⌬B2-Fc); wNg, wild-type noggin; IL-6R, hIL-6R␣-Fc.
a
This group was repeated (next line).
SD in italics.
All specimens were prepared for decalcified histology, cut- this tissue underwent membranous or metaplastic bone for-
ting them longitudinally, so that the ingrowth distance into mation. Because noggin is a highly specific BMP inhibi-
(2)
the chamber could be measured on histological slides by tor, our results therefore indicate that BMPs are necessary
computer-assisted morphometry. Three sections from each for membranous bone formation to occur, at least at a
specimen were measured and all were blinded and exam- normal rate.
ined in random order. The mean value from the three
The question then arises, if noggin effectively blocked
sections from each specimen was used for side comparison BMP activity, why was there any bone at all? We do not
using Students paired t-test.
RESULTS
know whether the noggin activity lasted for the entire ex-
perimental period or only for a part of it. Because there was
less bone with the noggin mutein at 4 weeks but not at 7
weeks, it appears that a single dose of noggin may not be
sufficient to counter the effects of endogenous BMPs over a
All groups with hNg⌬B2-Fc or wild-type noggin at 4
weeks showed decreased bone ingrowth (with one excep-
tion). This effect was seen regardless of dose and both in
comparison with buffer control and the IL-6R mutein (Table
7
-week time period. This may be caused by loss of noggin
from the chamber by a variety of mechanisms including
uptake and proteolysis or simple diffusion out of the cham-
ber or any combination thereof. The Fc chain on the noggin
molecule was of human origin, so it is possible that anti-
bodies formed and eliminated noggin activity during the
later part of the experiment. A less likely possibility would
be that a complete block of BMP signaling only leads to a
decreased rate of bone formation.
The IL-6R-Fc control shows that the hNg⌬B2-Fc did not
inhibit bone formation because of reactions to the human Fc
tag or by unspecific effects of the presence of microgram
amounts of recombinant protein, because if so, the IL-6R-Fc
should have inhibited bone formation as well. Further, such
unspecific effects probably should have an influence on the
total amount of ingrown tissue (bone and soft tissue) and the
histological appearance of the soft tissue, which was not
seen. The hNg⌬B2-Fc exhibits a prolonged half-life and
increased bioavailability on systemic administration and
was generated to enable treatment of certain conditions such
as fibrodysplasia ossificans progressiva (A.N. Economides,
X. Liu, F.H. Gannon, E.M. Shore, X. Wang, G. Elove, K.B.
Shriram, E.K. Fytros-Sirabian, J.P. Fandl, T.E. Ryan, K.M.
Bailey, F.S. Kaplan, R.B. Kimble, N. Stahl, unpublished
data, 2000). The effect of wild-type noggin in this experi-
ment shows that these modifications of the noggin molecule
were not needed in this model of local application.
1
; Fig. 2). At 7 weeks, there was no remaining effect. The
IL-6R-Fc showed no effect. The exception was the first
group with 10 ␮g hNg⌬B2-Fc versus NaCl, which for
unknown reasons showed an abnormal variability in bone
ingrowth and a decrease that was not significant. This ex-
periment was repeated and then showed inhibition by
hNg⌬B2-Fc (Table 1).
In none of the groups was there a significant side differ-
ence in total tissue ingrowth. Thus, the amount of tissue that
had entered the chamber was not affected by noggin. The
only change was in the extent to which this tissue had
differentiated to bone. Vehicle controls differed signifi-
cantly between groups.
The scintigraphic activities of the 10-␮g hNg⌬B2-Fc
repeat group showed less technetium uptake on the noggin
side in all rats but one (p ϭ 0.002), indicating less bone
formation. The technetium activity correlated with the bone
2
ingrowth distance (r ϭ 0.74 and p ϭ 0.001, control side
data only, to avoid dependence).
Five rats were excluded because of infection or technical
problems. All exclusions were done before the histological
evaluation.
DISCUSSION
Bone ingrowth generally was low in all groups compared
with what usually is seen at 6 weeks in this model. This
(6)
Our data indicate that noggin did not inhibited tissue probably is caused by both the shorter observation time and
ingrowth in the chamber, but did inhibit the extent to which a disturbance of ingrowth by the collagen carrier, which was

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ASPENBERG ET AL.
to a large extent unresorbed at 4 weeks. The carrier also
seemed to vary in density between different experimental
groups, probably depending on variation within and be-
tween the collagen sheets that the implanted pieces were cut
out from. This might explain the difference in control in-
growth between groups. The experiments were performed
one group at a time, but the experiment and control carrier
pieces for each group were prepared simultaneously each
time and chosen for treatment by random. Because of the
variation between groups, we have analyzed only paired
comparisons within groups with a less disturbing carrier,
noggin might have shown even greater effects.
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4
5
6
This study shows a physiological role for BMPs during
membranous bone formation in adult rats. Previously, ef-
fects of BMPs only have been shown by exogenous appli-
cation of BMPs, usually in pharmacologic doses. It is not
clear why such high doses of exogenous BMPs are required,
but increased expression of inhibitors such as noggin is one
(7)
possible explanation.
7
So far, a function of noggin during repair processes in
adult animals has not been shown, but considering the
similarities between skeletal development and repair, BMP
inhibitors may be important in fracture repair.
2
114.
Address reprint requests to:
Per Aspenberg
ACKNOWLEDGMENTS
The authors thank Inger Mårtensson and Carina Forslund
for technical assistance. This investigation was supported
by the Swedish Medical Research Council (project 2031),
The Medical Faculty of Lund, the King Gustaf V 80th
Department of Orthopedics
Lund University Hospital
221 85 Lund, Sweden
¨
Birthday Foundation, the Alfred Osterlund, and Greta och
Johan Kock foundations. Noggin was supplied by Regen- Received in original form June 28, 2000; in revised form Septem-
eron Pharmaceuticals, Inc. for a handling fee. ber 5, 2000; accepted October 18, 2000.