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Transforming growth factor-beta 3(Tgf-╬▓3) in a collagen gel delays fusion of the rat posterior interfrontal suture in vivo.

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THE ANATOMICAL RECORD 267:120 –130 (2002)
Transforming Growth Factor-Beta 3
(Tgf-␤3) in a Collagen Gel Delays
Fusion of the Rat Posterior
Interfrontal Suture In Vivo
Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M
University System Health Science Center, Dallas, Texas
Center for Craniofacial Research and Diagnosis, Baylor College of Dentistry, Texas
A&M University System Health Science Center, Dallas, Texas
Department of Pediatric Dentistry, College of Dentistry, Ohio State University,
Columbus, Ohio
Postnatal expansion of the intramembranous bones of the craniofacial skeleton occurs as bone growth at sutures.
Loss of the bone growth site occurs when the suture fails to form, or when the newly formed sutures become ossified,
resulting in premature obliteration. Previous experiments demonstrated that removal of dura mater from fetal rat
coronal sutures, or neutralizing transforming growth factor-beta 2 (Tgf-␤2) activity using antibodies resulted in
premature obliteration of the suture in vitro. Conversely, addition of Tgf-␤3 to coronal sutures in vitro rescued them
from osseous obliteration. To examine whether Tgf-␤3 rescues sutures from obliteration in vivo, a collagen gel was used
as a vehicle to deliver Tgf-␤3 to the normally fusing rat posterior interfrontal (IF) suture. Surgery was done on postnatal
day 9 (P9) rats, in which collagen gels containing 0, 3, or 30 ng Tgf-␤3 were placed above the IF suture, underneath the
periosteum for 2 weeks. By P24, 75–100% of animals in control unoperated, sham-operated, and collagen gel-only
groups had fused IF sutures. In contrast, 40% of sutures exposed to 3 ng Tgf-␤3 remained open, while sutures exposed
to 30 ng Tgf-␤ were similar to controls. By immunohistochemistry, sutures rescued from obliteration by Tgf-␤3 had the
same Tgf-␤ receptor type II (T␤r-II) distribution as controls. However, Tgf-␤3-treated sutures had altered Tgf-␤2 and
T␤r-I distribution compared to controls. Anat Rec 267:120 –130, 2002. © 2002 Wiley-Liss, Inc.
Key words: cranial suture morphogenesis; rat; collagen gel; Tgf-␤3; craniosynostosis
Development and growth of the calvaria is closely coordinated with the development and growth of the underlying brain (Kreiborg, 2000). The major sites of bone growth
in the calvaria are the sutures, which form when the
expanding bones of the craniofacial skeleton either butt
up against one another or overlap (Kokich, 1986). Failure
of sutures to form during embryonic development, or failure of newly formed sutures to remain unossified, results
in premature obliteration of sutures, termed craniosynostosis. This leads to loss of the bone growth site, often
resulting in severe facial dysmorphology (Enlow, 1986,
1989, 2000).
Several genetic mutations have been linked to craniosynostosis, including mutations in genes for fibroblast
growth factor receptors (FGFRs)1 (Jabs et al., 1994;
Muenke et al., 1994, 1997; Reardon et al., 1994; Rutland et
al., 1995; Wilkie et al., 1995; Bellus et al., 1996; ), MSX2
(Jabs et al., 1993), and TWIST (el Ghouzzi et al., 1997,
1999). There are no known mutations of transforming
growth factor betas (TGF-␤s) or their receptors (T␤R-I and
T␤R-II) that are associated with craniosynostosis. How©
Portions of this work were presented in abstract form at the
30th Annual Meeting of the American Association of Dental Research, Chicago, 2001.
Grant sponsor: NIH/NIDCR; Grant number: DE11978.
*Correspondence to: Lynne A. Opperman, Ph.D., Department of Biomedical Sciences, Baylor College of Dentistry, Texas
A&M University System Health Science Center, P.O. Box
660677, Dallas, TX 75266-0677. Fax: (214) 828-8951.
Received 12 December 2001; Accepted 5 March 2002
DOI 10.1002/ar.10094
Published online in Wiley InterScience
In line with new nomenclature, all abbreviations of genes will
be italicized, while all abbreviations of proteins will be regular
type. All abbreviations of human genes and proteins will be in
upper case, while abbreviations of animal genes and proteins will
have the first letter in upper case and all subsequent letters in
lower case.
ever, mutations in the TGF-␤-related NOGGIN are associated with proximal synphalangism and multiple synostoses syndrome, while mutations in the TGF-␤-related
CDMP1 are associated with Hunter Thompson-type acromesomelic chondrodysplasia and Grebe-type chondrodysplasia (see Massague et al., 2000, for review). More
importantly, mutations in the Smad transcriptional corepressor TGIF have been found to result in holoprosencephaly (Muenke and Beachy, 2000). In vitro data have
shown that Tgf-␤2 and Tgf-␤3 are potent regulators of
suture patency (Opperman et al., 1998, 1999, 2000).
Tgf-␤2 added to fetal rat calvaria in culture will induce the
normally patent coronal sutures to fuse, while Tgf-␤3 will
rescue these sutures from fusion in vitro. Because of this
antagonistic behavior, it is possible that the expression of
one of these proteins directly affects the expression or
presence of the other. Alternately, these factors could antagonistically affect expression of other factors or their
receptors involved in regulating suture patency. If so, the
relationship between Tgf-␤2 and Tgf-␤3 in regulating suture patency is of pathological and therapeutic importance. Manipulation of either Tgf-␤2 or Tgf-␤3 could be
used to regulate the activity of the other, or regulate
expression of other growth factors or their receptors whose
activity is affected by genetic mutations.
Prenatal diagnosis of craniosynostosis is difficult, and
intervention is not currently feasible. To correct the synostosis and preclude the deformational changes that occur
due to craniosynostosis, early postnatal surgical intervention is currently used (Marchac and Renier, 1982; Marsh
and Vannier, 1985; Persing et al., 1981, 1989; Jane and
Persing, 1986; Posnick, 1992, 1996a,b). A certain amount
of success has been achieved from the use of this technique; however, calvariae often show rapid bone growth
within the suture, leading to resynostosis. This necessitates further surgical intervention (Moss, 1959; Marchac
and Renier, 1982; Marsh and Vannier, 1985; Jane and
Persing, 1986; Ousterhout and Vargervik, 1987; Persing
et al., 1989; Hassler and Zentner, 1990; Fatah et al., 1992;
Drake et al., 1993). To avoid the problem of reossification,
many techniques have been employed, with varying success, to reduce the osteogenic potential of the calvaria
(Persing et al., 1989; Cohen, 1993; Salyer, 1999). However,
to date no soluble growth factors have been used to inhibit
reossification of the suture site in vivo.
To test the hypothesis that Tgf-␤3 plays a role in regulating suture patency in vivo, a well characterized model
utilizing the normally fusing posterior interfrontal (IF)
suture of rats was used to examine the ability of Tgf-␤3 to
regulate suture patency in vivo (Moss, 1960; Opperman et
al., 1997; Roth et al., 1997). A degradable collagen gel was
used as a vehicle for transporting and localizing Tgf-␤3
above IF sutures in young rats prior to the onset of suture
fusion. When the rats reached the age at which fusion
normally occurs, the sutures were examined for fusion.
Using immunohistochemical techniques, Tgf-␤2, T␤r-I,
and T␤r-II expression in Tgf-␤3-rescued IF sutures was
compared to their expression in fusing IF sutures and
nonfusing sagittal sutures. This was done to test the hypothesis that Tgf-␤3 rescues sutures by regulating expression of the receptors used by both Tgf-␤2 and Tgf-␤3,
rather than regulating Tgf-␤2 protein expression in sutures.
Preparation of Collagen Gels
A highly purified, bovine collagen type I gel prepared as
described by Rosenblatt et al. (1989), and generously provided by NeuColl Inc. (Campbell, CA), was used as a
carrier for keeping Tgf-␤3 localized above the suture region. Under sterile conditions, 100-␮l aliquots of the gel
(65 mg/ml) were extruded from a 1-ml syringe. Each aliquot was mixed with Tgf-␤3 to a final concentration of 0, 9,
or 90 ng per gel aliquot. The gel was then molded into a
3-mm-long cylinder and allowed to stand overnight in an
incubator at 37°C with 5% CO2 in air. Prior to insertion
into the animals, the gels were cut into 1-mm segments,
such that each segment contained 0, 3, or 30 ng Tgf-␤3.
One 1-mm-long segment was sufficient to cover the IF
suture of each animal.
Animals and Surgery
Thirty-three Sprague-Dawley rats were used in this
study. The protocol for surgery and postsurgical observation and care followed the guidelines dictated by the Baylor College of Dentistry IACUC. All animals for this study
were housed in the Animal Research Unit at the Baylor
College of Dentistry. The results of two separate experiments were combined for data analysis. Nine days after
birth (P9), rat pups were anesthetized using 3.25% isofluorane (Butler, Dallas, TX) and 900 cc/min O2 inhalant
anesthetic delivered by a Foregger F500 anesthesia system via a nose cone. A coronal incision through the scalp
was made from ear to ear, the skin was reflected to expose
the coronal and IF sutures, and the periosteum was loosened above the IF suture by use of a periosteal elevator.
Animals were randomly assigned to four groups. Group 1
was used as a sham surgical control. Animals in this group
had no further manipulation prior to closing the incision.
Group 2 had collagen gels with sterile phosphate-buffered
saline (PBS) placed on the exposed IF suture, beneath the
periosteum. Group 3 received collagen gels with 3 ng Tgf␤3, and group 4 received collagen gels with 30 ng Tgf-␤3.
The two edges of the scalp were reapproximated and
closed using 6-0 silk suture (DG, New York, NY), without
damage or movement of the gel.
Fifteen days after surgery (P24), all rats from the four
groups were killed by Halothane (Butler) overdose. Group
5 comprised pups killed at P24, which served as nonsurgical controls. All animals were decapitated and the scalps
were removed. The heads were fixed overnight in 4% paraformaldehyde and decalcified for 7 days with 0.5 M EDTA.
Following decalcification, the heads were cut along the
coronal suture, separating each into cranial regions containing frontal bones with intervening IF sutures and
parietal bones with intervening sagittal sutures. Samples
were post-fixed overnight in 4% paraformaldehyde and
processed for paraffin embedding and sectioning. Finally,
6-␮m sections through tissues containing either IF or sagittal sutures were cut and stained with hematoxylin and
eosin (H&E) or used for immunohistochemical analysis.
An indirect immunoperoxidase procedure was carried
out as described previously (Opperman et al., 1997).
Briefly, endogenous peroxidase activity was removed by
pre-incubation in 3% hydrogen peroxide in methanol. PBS
containing 2% normal rabbit serum (Sigma, St. Louis,
TABLE 1. Results of histological analysis of interfrontal suture patency
Total sample
Sham surgical control
Collagen gel only
Gel with 3ng TGF-␤3
Gel with 30ng TGF-␤3
Non-surgical control
MO) was used to block nonspecific binding of secondary
antibody. Primary polyclonal antibodies to Tgf-␤2 (R & D
Systems, Minneapolis, MN), T␤r-I, or T␤r-II (Santa Cruz
Biotechnologies, Santa Cruz, CA), company-tested for
specificity by western blotting procedures, were used at a
1:250 concentration. Control slides were incubated in the
absence of primary antibody. Secondary antibodies were
peroxidase-conjugated rabbit anti-goat IgG (1:250; Jackson Immunoresearch Laboratories, West Grove, PA) with
diaminobenzidine kit (Vector Laboratories, Burlingame,
CA) as chromagen.
Scoring and Analysis of Tissues
Depending on the suitability of the tissue for scoring,
between 24 and 36 sections through the mid-section of the
sutures were examined. Sections through sutures were
scored as open or fused by two observers blinded to tissue
grouping. Suture patency was scored as fused or open
according to the presence or absence of bony bridges
across the suture matrix. Sutures were designated as open
if less than 10% of sections had bony bridging, while
sutures with greater than 10% of sections with bony
bridges across sutures were scored as fused. Sagittal sutures, which remain open throughout the life of the animal, were used as control sutures.
Tgf-␤2 immunoreactivity in sagittal sutures was scored
as absent, light, or dark. Immunoreactivity in IF suture
tissues was scored as either lighter or darker than Tgf-␤2
immunoreactivity in sagittal sutures. T␤r-I or T␤r-II immunoreactivity was scored as present in most (⬎70%)
cells, or present in few (⬍30%) cells within sutural tissues.
No tissues had populations of cells with a mixture of
immunoreactive and nonimmunoreactive cells in the
range of 30 –70% for either T␤r-I or T␤r-II immunoreactivity.
Effect of Tgf-␤3 on IF Suture Patency In Vivo
IF and sagittal sutures were examined. Sagittal and IF
sutures are butt sutures, with bone fronts that overlap
slightly but never become as interdigitated or complex as
the overlapping coronal and lambdoid sutures. Sagittal
sutures remain patent throughout the life of the rat, and
in all nonsurgical control animals the sagittal suture remained patent (Table 1). The suture was visible as fibrous,
cellular tissue between the parietal bones (Fig. 1A and B).
One animal in each of groups 1, 2, and 3 had sagittal
sutures in which more than 10% of sections through the
suture were scored as fused. A bony bridge between the
parietal bones was seen spanning the suture (Fig. 1C and
D). The remaining animals in each group had open sagittal sutures, similar to controls shown in Figure 1A and B.
All sagittal sutures in group 4 animals were scored as
open, similar to unoperated controls (not shown).
All IF sutures in group 5 animals were fused (Table 1).
The bone fronts on either side of the IF suture appeared
thickened compared to the bone further away from the
suture region (Fig. 2A and B). Suture fusion appeared as
a complete bridging of the frontal bones across the dural
surface, with residual suture matrix visible above the
fused bones. In control groups 1 and 2, the majority of
animals (67% and 80%, respectively) had bony bridges
across the suture (Fig. 2C and D) or showed typical fusion
across the dural surface, similar to that seen in nonsurgical controls.
In group 3, 60% of the animals had sutures that were
rescued from obliteration (Table 1). The rescued sutures
appeared widely patent, with the bone fronts separated by
a fibrous, cellular matrix similar to that seen in normal
sagittal sutures (Fig. 3A and D). Those sutures that were
not rescued varied from partial obliteration of the suture
by bony bridging (Fig. 3B and E) to complete fusion, as in
untreated controls (not shown). The majority of sutures in
group 4 were not rescued from fusion (Table 1). In those
that did remain patent, the bone fronts were thicker than
bones in untreated sutures, and the suture was markedly
narrowed (Fig. 3C and F). The small sample size precluded a statistical analysis of the data.
Effect of Tgf-␤3 on Tgf-␤2 Immunoreactivity in
IF Sutures
Sagittal sutures, used as normal nonfusing controls,
showed little to no immunoreactivity for Tgf-␤2 within the
cells or matrix of the suture proper, but showed intense
levels of Tgf-␤2 immunoreactivity in osteoblasts lining the
bone fronts on either side of the suture (Fig. 4A and E).
Similarly, fusing IF sutures showed intense levels of
Tgf-␤2 immunoreactivity within the osteoblasts lining the
bone fronts of the sutures, and in the cells and matrix of
the surrounding periosteum and dura mater (Fig. 4B and
F). In contrast to sagittal sutures, the residual IF suture
matrices and cells were highly immunoreactive for Tgf-␤2.
Sutures rescued from obliteration by Tgf-␤3 were found to
have strong immunoreactivity for Tgf-␤2 in the osteoblasts lining the bone fronts, similar to that seen in both
fusing IF sutures and patent sagittal sutures (Fig. 4C and
G). However, immunoreactivity within the matrix and
cells of sutures rescued from obliteration by Tgf-␤3 was
low to absent, similar to that seen in nonfusing sagittal
sutures, and in contrast to that seen in fusing IF sutures.
Sections used as immunohistochemical controls and that
were not exposed to primary antibodies showed no immunoreactivity in any tissues (Fig. 4D and H).
Fig. 1. Micrographs of H&E-stained sections through the sagittal
sutures of P24 rats. A: Low-power image showing parietal bones (pb) on
either side of the fibrous suture (s) are thicker close up to the suture and
appear narrower distal to the suture site. B: High-power image of the
suture region. C: Low-power image through the fused sagittal suture,
showing greatly thickened bones. D: High-power image showing residual suture (asterisk) and fusion of parietal bones across the suture
(arrow). d, dura mater, p, periosteum. Original magnification: (A and C)
20⫻, bar ⫽ 30 ␮m; (B and D) 40⫻, bar ⫽ 60 ␮m.
Fig. 2. Micrographs of H&E-stained sections through IF sutures of
P24 rats. A and B: Low- and high-power images of a nonsurgical control
suture, showing residual suture matrix (asterisk) above the fused frontal
bones (arrow). C and D: Low- and high-power images of a fused suture
in a gel-only treated animal, showing a bony bridge through the center of
the suture (arrow). Original magnification: (A and C) 10⫻, bar ⫽ 15 ␮m;
(B, and D) 20⫻, bar ⫽ 30 ␮m.
Effect of Tgf-␤3 on T␤r-II Immunoreactivity in
IF Sutures
surfaces of the bone away from the suture site. In contrast,
periosteal fibroblasts were nonreactive for T␤r-II in all
groups. In nonfusing sagittal sutures, most of the osteoblastic cells lining the bone fronts on either side of the
In all groups, T␤r-II immunoreactivity was present in
most of the osteoblasts lining the dural and periosteal
Fig. 3. Micrographs of H&E-stained sections through IF sutures of
P24 rats. A and D: Low- and high-power images through a suture treated
with 3 ng Tgf-␤3. The suture (s) remains widely patent between frontal
bones (fb). The suture can be seen to be closely associated with and
indistinguishable from the underlying dura mater (d) and overlying periosteum (p). B and E: Low- and high-power images of a suture treated
with 3 ng Tgf-␤3, in which the suture fails to remain patent. Small bony
bridges occur across the dural surface of the suture (arrow), while the
remainder of the suture remains widely patent. C and F: Low- and
high-power images through a suture treated with 30 ng Tgf-␤3. Highly
thickened frontal bones (fb) are separated by a narrow but patent suture
(s). Original magnification: (A–C) 10⫻, bar ⫽ 15 ␮m; (D–F) 20⫻, bar ⫽
30 ␮m.
suture and most of the fibroblastic cells within the suture matrix were immunoreactive for T␤r-II (Fig. 5A
and D). Similarly, in both fusing IF sutures (Fig. 5B and
E) and IF sutures rescued from obliteration by Tgf-␤3
(Fig. 5C and F), T␤r-II immunoreactivity was noted in
most of the osteoblastic cells lining the bone fronts, and
in most fibroblastic cells within the matrix of the sutures.
Effect of Tgf-␤3 on Intensity of T␤r-I
Immunoreactivity in IF Sutures
Similar to the immunoreactivity seen for T␤r-II, most of
the osteoblasts lining the dural and periosteal surfaces of
the bone, as well as those lining the bone fronts on either
side of the suture, were immunoreactive for T␤r-I in all
groups. Fibroblasts within the periosteum above the su-
Fig. 4. Micrographs of immunoreactivity for Tgf-␤2 in P24 rat (A and
E) sagittal and (B–H) IF sutures. A and E: Low- and high-power micrographs of the nonfusing sagittal suture. Tgf-␤2 immunopositive cells can
be seen lining the bone fronts on either side of the suture (large arrows).
Most of the suture cells within the suture matrix are negative for Tgf-␤2
immunoreactivity (small arrows). B and F: Low- and high-power micrographs of normally fusing IF sutures. Osteoblastic cells lining the bone
fronts are immunoreactive for Tgf-␤2 (large arrows), as are cells within
the sutures (small arrows), along with the suture matrix. C and G: Low-
and high-power micrographs of an IF suture rescued from obliteration by
Tgf-␤2. Immunoreactive osteoblasts can be seen lining the bone fronts
(large arrows), with nonreactive cells within the suture matrix (small
arrows). D and H: Low- and high-power micrographs of a sagittal suture
used as a negative control. Note complete absence of immunoreactivity
in osteoblasts (large arrows) and suture cells (small arrows), as well as in
the matrix. Original magnification: (A–D) 10⫻, bar ⫽ 15 ␮m; (E–H) 80⫻,
bar ⫽ 120 ␮m.
Fig. 5. Micrographs of immunoreactivity for T␤r-II in sections through
the (A and D) sagittal and (B–F) IF sutures of P24 rats. A and D: Low- and
high-power micrographs of sections through sagittal sutures. Darkly
immunoreactive osteoblasts can be seen lining the bone fronts on either
side of the patent sagittal suture (large arrows). Most of the fibroblastic
cells within the suture matrix are immunoreactive for T␤r-II (small arrows). B and E: Low- and high-power micrographs through fusing IF
sutures. Frontal bones can be seen to be fused underneath the residual
suture. Osteoblasts lining the residual suture are highly immunoreactive
for T␤r-II (large arrows), as are fibroblastic cells within the suture matrix
(small arrows). C and F: Low- and high-power micrographs of sutures
rescued from obliteration by 3 ng Tgf-␤3. Note immunoreactivity for
T␤r-II in the osteoblasts lining the bone (large arrows) and in the fibroblastic cells within the suture matrix (small arrows). Original magnification: (A–C) 30⫻, bar ⫽ 45 ␮m; (D–F) 80⫻, bar ⫽ 120 ␮m.
Fig. 6. Micrographs of immunoreactivity for T␤r-I in sections through
the (A and D) sagittal and (B–F) IF sutures of P24 rats. A and D: Low- and
high-power micrographs of sections through sagittal sutures. Immunoreactive osteoblasts (large arrows) can be seen around the leading
edges of bone fronts on either side of the suture. Suture fibroblasts
(small arrows) are not immunoreactive. B and E: Low- and high-power
micrographs of sections through fusing IF sutures. Note immunoreactive
osteoblasts (large arrows) lining the bone fronts, and immunoreactive
suture fibroblasts (small arrows) within the residual suture matrix. C and
F: Low- and high-power micrographs of sections through IF sutures
rescued from fusion by 3 ng Tgf-␤3. Note a distribution of osteoblasts
(large arrows) similar to those seen in nonfusing sagittal sutures (described in A and D). Within the suture matrix, note suture fibroblasts
(small arrows) that lack T␤r-I immunoreactivity. Original magnification:
(A–C) 30⫻, bar ⫽ 45 ␮m; (D–F) 80⫻, bar ⫽ 120 ␮m.
tures were negative for T␤r-I immunoreactivity in all
groups, similar to that seen for T␤r-II immunoreactivity.
Most suture fibroblasts within the matrix of nonfusing
sagittal sutures were negative for T␤r-I immunoreactivity
(Fig. 6A and D). In contrast, all fibroblastic cells within
the residual matrix of fusing IF sutures were immunoreactive for T␤r-I (Fig. 6B and E). Unlike fibroblasts within
fusing IF sutures, but similar to those in nonfusing sagit-
tal sutures, fibroblasts in IF sutures rescued from obliteration by Tgf-␤3 were not immunoreactive for T␤r-I (Fig.
6C and F).
In vitro, Bmp2, Bmp4 (Kim et al., 1998), and Tgf-␤2
(Opperman et al., 1997) have been shown to induce suture
fusion. Furthermore, Tgf-␤3 (Opperman et al., 2000) and
neutralizing antibodies to Tgf-␤2 (Opperman et al., 1999)
rescued sutures from fusion in vitro. In vivo, Tgf-␤2
(Roth et al., 1997), and Fgf2 (Iseki et al., 1999) have both
been reported to induce or accelerate suture fusion. To
date, however, no soluble factors have been shown to
inhibit reossification of the suture site in vivo. The potential of using growth factors to inhibit reossification of the
suture in conjunction with current initial surgery, or, preferably, with more limited and localized surgery, has great
importance for reducing surgical risk and improving outcome.
In the rat, all of the cranial sutures remain patent
throughout the life of the animal, with the exception of
the posterior region of the IF suture (Moss, 1954). This
region of the suture undergoes osseous obliteration early,
becoming completely obliterated by P21 (Moss, 1960). This
pattern of early postnatal fusion is similar to that found
in the metopic suture in humans, which becomes obliterated by 2–3 years after birth (Cohen, 2000). The rat IF
suture, as a model of naturally occurring suture fusion,
was used to test whether Tgf-␤3 can prevent or delay
suture fusion.
A low dose of Tgf-␤3 rescued IF sutures from obliteration in vivo, although not all IF sutures in animals from
the low-dose group were rescued. A high dose of Tgf-␤3
failed to rescue sutures from obliteration, similar to vehicle alone or sham operation. These findings are similar to
those reported in vitro (Opperman et al., 2000). Tgf-␤3 is
known to be a biphasic regulator of osteoblast activity,
with lower concentrations of Tgf-␤3 stimulating cell proliferation, while higher concentrations inhibit cell proliferation and stimulate production of extracellular matrix
and markers of cellular differentiation (ten Dijke et al.,
1990). It is therefore possible that the higher dose of
Tgf-␤3, which did not rescue sutures from obliteration,
instead stimulated osteogenesis, resulting in suture obliteration. This is not easy to demonstrate, as these sutures
are destined to fuse, so markers of osteoblast differentiation would be present both in the absence of Tgf-␤3 and in
the presence of the high dose of Tgf-␤3. One way to address this problem would be to place gels containing low
and high doses of Tgf-␤3 above the sagittal suture, which
remains patent. If the high dose of Tgf-␤3 induced fusion
of the sagittal sutures, then it would be interesting to look
for markers of osteoblast differentiation within these fusing sutures.
Some fusion of sagittal sutures from animals undergoing sham and vehicle-only procedures was noted, suggesting that the surgical procedure may result in sporadic
bony bridging within the suture. In support of this idea, it
has been noted anecdotally in clinical cases (Genecov,
personal communication) that intermittent bony bridging
within sutures can occur in association with surgical procedures. Alternately, since collagen gels were used as a
vehicle, it is possible that most of the Tgf-␤3 was released
from the gels early and hence the rescue effect of Tgf-␤3
disappeared, with the sutures reentering the fusion process. However, since most of the sutures remained unfused, this is unlikely.
Various types of collagen biomaterials have been tested
as drug delivery vehicles (Friess, 1998), and delivery of
growth factors using this particular gel has been evaluated (Schroeder-Tefft et al., 1997; Bentz et al., 1998). In a
mink lung cell bioassay, Tgf-␤2 admixed with gel retained
a level of activity similar to that of Tgf-␤2 alone (Bentz et
al., 1998; Schroeder-Tefft et al., 1997). In vivo, Tgf-␤2 in
collagen gel induced a typical subcutaneous fibroblastic
response, indicating that the growth factor retained its
biological activity in the gel (Schroeder-Tefft et al., 1997).
Although highly purified, this formulation retains the native fibrillar conformation of collagen, as opposed to other
commercially available degraded collagen gels. The antigenic N- and C-terminal domains have been removed,
greatly reducing the likelihood of an immune response
(DeLustro et al., 1986; Ellingsworth et al., 1986).
There are a variety of clinical applications for this collagen gel. It is most widely used as an intradermal injection for soft-tissue augmentation, in which it resorbs in
approximately 6 months to a year (Pachence, 1996). Its
retention at other sites and in other species is not well
known. However, studies by Moursi and coworkers (unpublished data) indicated that biotinylated collagen was
visualized histologically up to 63 days postimplantation in
vivo at cranial sutures in rabbits. There was no evidence
of an inflammatory response, immune reaction, or fibrosis.
In addition, the injectable material is semiviscous at room
temperature and gels at 37°C, making it ideal for placement into surgical sites. Together these studies support
the use of this collagen gel as a vehicle for the delivery of
growth factors to cranial sutures.
Before growth factors can be used therapeutically for
rescuing sutures from obliteration, it is important to understand the mechanism by which growth factors regulate
suture patency. Tgf-␤3 reduced Tgf-␤2 immunoreactivity
in matrix and cells of IF sutures when compared to untreated IF sutures, with immunoreactivity appearing
more similar to that of nonfusing sagittal sutures. This
finding is different from the effect seen in vitro, wherein
Tgf-␤2 immunoreactivity was unchanged in response to
Tgf-␤3 treatment, remaining as intense in the suture matrix of Tgf-␤3-rescued sutures as it was in fusing sutures
(Opperman et al., 2000). The difference between the in
vivo and the in vitro effects could be related to the fact that
the tissue culture environment is different from that
found in vivo. However, this difference demonstrates that
Tgf-␤3-induced changes in Tgf-␤2 immunoreactivity are
probably not primarily responsible for the ability of Tgf-␤3
to rescue sutures from fusion in vivo.
It is important in any study examining growth factor
effects to determine whether receptors for the growth factors are present, and whether the cells that express them
respond to the growth factors. Both T␤r-I and T␤r-II receptors are present on suture fibroblasts and osteoblasts.
No change in the number of T␤r-II immunoreactive cells
in sutures was found in response to Tgf-␤3 treatment.
This does not preclude a change in the number of receptors per cell; however, immunoreactivity was not considered sensitive enough to gauge differences in receptor
numbers per cell. In contrast, Tgf-␤3 dramatically reduced
the numbers of suture fibroblasts immunoreactive for
T␤r-I in IF sutures rescued from obliteration, when com-
pared to fusing IF sutures. This is similar to the findings
of Opperman et al. (2002), who reported down-regulation
of T␤r-I in IF suture fibroblasts in vitro in response to
Tgf-␤3 treatment. Interestingly, Opperman et al. (2002)
reported that nonfusing coronal sutures had many T␤r-Ipositive fibroblasts and osteoblasts within the suture,
whereas the data presented here indicate that nonfusing
sagittal sutures have few T␤r-I immunoreactive fibroblasts. This may reflect developmental differences between these sutures, which are also shown to have differences in Msx2 and Shh expression (Liu et al., 1995; Kim et
al., 1998).
While it has been shown that Tgf-␤2 and Tgf-␤3 have
antagonistic effects in regulating suture morphogenesis
(Opperman et al., 2000), they have been shown to have
distinctly different sequential regulatory effects during
epithelial to mesenchymal transformation in embryonic
heart (Boyer et al., 1999), and differential effects on medial edge epithelium during fusion of the palatal shelves
(Taya et al., 1999). It is intriguing that Tgf-␤2 and Tgf-␤3
have such opposite effects on cell function, when they
share the same set of transmembrane receptors and the
same intracellular Smads (Smad2, Smad3, and perhaps
Smad5 (Centrella et al., 1996)). These Smads interact
with the ubiquitous Smad4 after Tgf-␤ receptor stimulation and then translocate to the nucleus, where they interact with other transcription factors such as SP1 (Inagaki et al., 2001; Poncelet and Schnaper, 2001), c-Jun/cFos (Zhang et al., 1998), and Runx2 (Leboy et al., 2001) to
activate Tgf-␤-inducible transcription (Massague and
Wotton, 2000). Of note, it now appears that Tgf-␤ signaling may interact with the MAP kinase signaling pathways
of tyrosine kinase receptors. Tgf-␤ can signal via the Ras/
Mek pathway to phosphorylate Smad1 (Yue et al., 1999),
Tgf-␤1 activation of Atf-2 is dependent upon Smad4 activation of p38 (Simeone et al., 2001), and blocking the
Mek1/Erk pathway inhibits Tgf-␤1-induced ␣1(I) collagen
expression (Hayashida et al., 1999). Furthermore, there is
now good evidence that Tgf-␤s can signal through these
other pathways, independently of Smads. For example,
they can activate Jnk signaling in a Rho-dependent,
Smad-independent manner to activate Ap-1 (Engel et al.,
1999), c-Jun and Atf-2 function (Hocevar et al., 1999;
Rousse et al., 2001), and mobilization of the actin cytoskeleton (Edlund et al., in press), suggesting that alternate
Tgf-␤ signaling pathways from the Smad pathway exist.
This could account for differences in cell function seen in
response to Tgf-␤1, Tgf-␤2, and Tgf-␤3 during suture (Opperman et al., 1999, 2000), palate (Taya et al., 1999), and
heart morphogenesis (Boyer et al., 1999).
While different Tgf-␤s can have distinct effects using
the same receptors, it appears that different Tgf-␤ receptors can also have distinct activities in response to growth
factor binding (Boyer and Runyan, 2001). This means that
Tgf-␤s likely can regulate the activity of each other, either
by regulating different signaling pathways or by regulating access to the Tgf-␤ receptors.
It is now apparent that Tgf-␤3 can regulate suture
patency in vivo, maintaining the suture in its unossified state. However, it is of critical importance to determine the long-term effects of Tgf-␤3 on suture patency and
to determine whether high doses of Tgf-␤3 are deleterious
to nonfusing sutures. It is also necessary to develop a
better understanding of how Tgf-␤s interact with one
another and their receptors to regulate developing su-
tures, before these factors can be considered to be clinically useful.
The authors thank Mr. Phillip Winnard for advice concerning the technical aspects of gel preparation, and Mr. Greg
Cooper for doing the surgical procedures and tissue harvesting.
We also thank Drs. J. Pascal Mbiene, Kathy K. Svoboda, and
Bob Hinton, and Mr. Joseph T. Rawlins for critical reading of
the manuscript, and NeuColl Inc. (Campbell, CA) for generously donating the gel used in the experiments.
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