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Rescue of coronal suture fusion using transforming growth factor-beta 3 (Tgf-╬▓3) in rabbits with delayed-onset craniosynostosis.

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THE ANATOMICAL RECORD PART A 274A:962–971 (2003)
Rescue of Coronal Suture Fusion
Using Transforming Growth FactorBeta 3 (Tgf-␤3) in Rabbits With
Delayed-Onset Craniosynostosis
SHERRI LYN CHONG,1 RONAL MITCHELL,1 AMR M. MOURSI,2
PHILLIP WINNARD,2 H. WOLFGANG LOSKEN,3,4 JAMES BRADLEY,4
OMER R. OZERDEM,4 KODI AZARI,4 OGUZ ACARTURK,4
LYNNE A. OPPERMAN,5 MICHAEL I. SIEGEL,6,7 AND
MARK P. MOONEY1,4,6,7*
1
Department of Oral Medicine and Pathology, School of Dental Medicine, University
of Pittsburgh, Pittsburgh, Pennsylvania
2
Department of Pediatric Dentistry, College of Dentistry, Ohio State University,
Columbus, Ohio
3
Department of Plastic Surgery, School of Medicine, University of North Carolina,
Chapel Hill, North Carolina
4
Department of Plastic Surgery, School of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania
5
Department of Biomedical Sciences, Baylor College of Dentistry, Texas A & M
University System Health Center, Dallas, Texas
6
Department of Anthropology, University of Pittsburgh, Pittsburgh, Pennsylvania
7
Department of Orthodontics, University of Pittsburgh, Pittsburgh, Pennsylvania
ABSTRACT
Craniosynostosis results in cranial deformities and increased intracranial pressure, which pose extensive and
recurrent surgical management problems. Developmental studies in rodents have shown that low levels of transforming
growth factor-␤3 (Tgf-␤3) are associated with normal fusion of the interfrontal (IF) suture, and that Tgf-␤3 prevents IF
suture fusion in a dose-dependent fashion. The present study was designed to test the hypothesis that Tgf-␤3 can also
prevent or “rescue” fusing sutures in a rabbit model with familial craniosynostosis. One hundred coronal sutures from
50 rabbits with delayed-onset, coronal suture synostosis were examined in the present study. The rabbits were divided
into five groups of 10 rabbits each: 1) sham controls, 2) bovine serum albumin (BSA, 500 ng) low-dose protein controls,
3) low-dose Tgf-␤3 (500 ng), 4) high-dose BSA (1,000 ng) controls, and 5) high-dose Tgf-␤3 (1,000 ng). At 10 days of age,
radiopaque amalgam markers were implanted in all of the rabbits on either side of the coronal suture to monitor sutural
growth. At 25 days of age, the BSA or Tgf-␤3 was combined with a slow-absorbing collagen vehicle and injected
subperiosteally above the coronal suture. Radiographic results revealed that high-dose Tgf-␤3 rabbits had significantly
greater (P ⬍ 0.05) coronal suture marker separation than the other groups. Histomorphometric analysis revealed that
high-dose Tgf-␤3 rabbits also had patent coronal sutures and significantly (P ⬍ 0.01) greater sutural widths and areas
than the other groups. The results suggest that there is a dose-dependent effect of TGF-␤3 on suture morphology and
area in these rabbits, and that the manipulation of such growth factors may have clinical applications in the treatment
of craniosynostosis. Anat Rec Part A 274A:962–971, 2003. © 2003 Wiley-Liss, Inc.
Key words: rabbit; craniosynostosis; Tgf-␤3; coronal suture; cytokine therapy
Presented in part at the annual meetings of the 80th International Association for Dental Research, San Diego, 2002; the 59th
American Cleft Palate-Craniofacial Association, Seattle, 2002;
and the Plastic Surgery Research Council, Boston, 2002.
Grant sponsor: NIH/NIDCR; Grant numbers: DE13078;
DE07336; Grant sponsor: NIH/NIAMS; Grant number: AR46382;
Grant sponsor: Oral and Maxillofacial Surgery Foundation;
Grant sponsor: Children’s Hospital of Pittsburgh.
©
2003 WILEY-LISS, INC.
*Correspondence to: Mark P. Mooney, Ph.D., Department
of Oral Medicine and Pathology, 329 Salk Hall, University
of Pittsburgh, Pittsburgh, PA 15261. Fax: (412) 648-7535.
E-mail: mpm4@pitt.edu
Received 19 March 2003; Accepted 9 July 2003
DOI 10.1002/ar.a.10113
SUTURE FUSION RESCUE USING Tgf-␤3
The birth prevalence of simple, nonsyndromic craniosynostosis has been estimated at 300 –500 per 1,000,000
births (Cohen, 1979, 1989; Cohen and Kreiborg, 1992).
Premature coronal suture synostosis is associated with
secondary deformities in the cranial vault and cranial
base (Babler and Persing, 1982; Marsh and Vannier, 1985,
1986; Burdi et al., 1986; Babler, 1989; Hoyte, 1989;
Mooney et al., 1994b; Burrows et al., 1995; Smith et al.,
1996), significantly elevated intracranial pressure (Renier, 1989; Gault et al., 1992; Mooney et al., 1998a, 1999;
Fellows-Mayle et al., 2000), and altered intracranial volume (Singhal et al., 1997; Hudgins et al., 1998; Mooney et
al., 1998a,b; Camfield et al., 2000). These conditions may
result in optic nerve compression and papilledema, and, if
left uncorrected, optic atrophy, blindness (Miller, 2000),
cognitive disabilities, and mental retardation (Kapp-Simmonds et al., 1993; Arnaud et al., 1995; Camfield et al.,
2000; Persing and Jane, 2000). Such severe craniofacial
growth, ocular, and neural abnormalities pose extensive,
costly, and often recurrent clinical and surgical management problems (Marchac and Renier, 1982; Marsh and
Vannier, 1985; Ousterhout and Vargervik, 1987; Persing
et al., 1989; Fatah et al., 1992; Dufresne and Richtsmeier,
1995; Turvey et al., 1996; Williams et al., 1997; Persing
and Jane, 2000; Posnick, 2000). In addition, the surgical
sites often show excessive reossification and resynostosis,
which require additional surgical procedures and increase
patient morbidity and mortality (Marchac and Renier,
1982; Marsh and Vannier, 1985; Ousterhout and Vargervik, 1987; Persing et al., 1989; Fatah et al., 1992; Dufresne
and Richtsmeier, 1995; Turvey et al., 1996; Williams et al.,
1997; Persing and Jane, 2000; Posnick, 2000).
While a number of advances have been made in identifying the genetic etiologies of various craniosynostotic
syndromes (Cohen, 2000a; Jabs, 2002), the pathogenesis
of this condition is still not completely understood (Cohen,
2000b; Opperman and Ogle, 2002). However, recent work
has shown that differential expression of various transforming growth factor-␤ (Tgf-␤) isoforms plays a crucial
role in regulating suture patency once the sutures have
formed (Opperman et al., 1997, 1999, 2000; Roth et al.,
1997a,b; Cohen, 2000c; Opperman and Ogle, 2002), and
may also play a role in craniosynostosis (Cohen, 2000c;
Opperman and Ogle, 2002). All three Tgf-␤s are present in
the dura mater and in the osteoblasts lining the dural and
periosteal surfaces of the cranial bones after the suture is
fully formed (Opperman et al., 1997; Roth et al., 1997b);
however, they have different distributions within the suture matrix and bone fronts during obliteration and initial
suture formation (Opperman and Ogle, 2002). While there
are little to no Tgf-␤s in the suture matrix during initial
suture formation and in patent sutures, the suture matrix
of fusing sutures contains high levels of Tgf-␤1 and Tgf-␤2
(Opperman et al., 1997; Roth et al., 1997a,b; Most et al.,
1998). The osteogenic bone fronts on either side of the
suture contain Tgf-␤1 and Tgf-␤3 during initial suture
formation, and all three Tgf-␤s are present in the bone
fronts of fully formed sutures. However, Tgf-␤3 is absent
in the bone fronts of fusing sutures (Opperman et al.,
1997; Roth et al., 1997b). Similar findings have also been
obtained under craniosynostotic conditions. Differential
expression of Tgf-␤ isoforms have been observed in the
perisutural tissues of human infants (Lin et al., 1997;
Roth et al., 1997b) and synostotic rabbits (Poisson et al.,
1999). In addition, recent studies have shown that Tgf-␤3
963
will rescue normally fusing rodent sutures both in vitro
(Opperman et al., 1999; 2000) and in vivo (Opperman et
al., 2002a,b), probably via altered Tgf-␤2 expression and
T␤r-I distribution in the suture (Opperman et al., 2002a,
b). It has been suggested that the manipulation of these
soluble growth factors may have clinical applications for
preventing initial suture fusion (i.e., craniosynostosis) and
inhibiting postoperative reossification (Roth et al., 1997b;
Opperman et al., 1999, 2000, 2002b; Chong et al., 2001,
2002; Mooney et al., 2002a; Opperman and Ogle, 2002).
The present study was designed to test this hypothesis
in a strain of rabbits with familial craniosynostosis
(Mooney et al., 1994a, 1996a, 1998c). In particular, a number of clinical (Reddy et al., 1990; Hoffman and Reddy,
1991; Cohen et al., 1993; Cohen and MacLean, 2000) and
animal (Mooney et al., 1994a, 1996a, 2002b; Burrows et
al., 1995; Losken et al., 1998, 1999) studies have identified
and described a subset of craniosynostotic individuals
with familial, nonsyndromic, delayed-onset (i.e., postgestational) craniosynostosis. Although the pathogenesis of
delayed-onset or progressive synostosis is not known, it
has been suggested that this condition may be a variable
phenotypic expression of a familial craniosynostotic condition, and may represent part of a synostotic continuum
(Reddy et al., 1990; Hoffman and Reddy, 1991; Cohen et
al., 1993; Mooney et al., 1994a, 1996a, 2002; Losken et al.,
1998, 1999). While the suture is not completely synostosed
in craniosynostotic individuals, dense collagen bundles
and small bony bridges have been observed crossing the
suture (Mooney et al., 1996a,b; Losken et al., 1999). These
structures could effectively immobilize the suture, and
result in growth restrictions and altered brain growth
vectors. If individuals with delayed-onset synostosis have
not yet exhibited complete suture fusion, then exposing
the sutures to Tgf-␤3 in a slow-degrading vehicle may
prevent or “rescue” craniosynostosis. The present study
was designed to test this hypothesis in a rabbit model with
naturally occurring delayed-onset coronal suture synostosis.
MATERIALS AND METHODS
Sample
One hundred sutures from 50 10-day-old New Zealand
White rabbits (Oryctolagus cuniculus) were examined in
the present study (Fig. 1). All of the rabbits were born in
our ongoing breeding colony of congenitally synostosed
rabbits at the Department of Anthropology vivarium, University of Pittsburgh. The breeding colony has been well
documented, and systematic treatment and data-collection protocols have been followed (Mooney et al., 1994a,
1996a,b, 1998a). This study was reviewed and approved
by the University of Pittsburgh Institutional Animal Care
and Use Committee (IACUC).
The rabbits were randomly assigned to five groups of 10
rabbits each, as follows: 1) a sham group, which functioned as a surgical control suture group; 2) a low-dose,
protein control group: sutures treated with 500 ng/suture
of bovine serum albumin (BSA); 3) a low-dose treatment
group: sutures treated with 500 ng/suture of Tgf-␤3; 4) a
high-dose, protein control group: sutures treated with
1,000 ng/suture of BSA; and 5) a high-dose treatment
group: sutures treated with 1,000 ng/suture of Tgf-␤3.
There were 20 sutures in each group.
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CHONG ET AL.
Fig. 1. Cleaned and dried skulls and coronal sutures from a 42-day-old wild-type rabbit and a rabbit with
delayed-onset craniosynostosis. Note the dysmorphic coronal suture in the delayed-onset synostotic rabbit.
Surgery
At 10 days of age, the rabbits were anesthetized with an
IM injection (0.59 ml/kg) of a solution of 91% Ketaset
(ketamine hydrochloride, 100 mg/ml) and 9% Rompun (xylazine, 20 mg/ml). The scalps were then shaved, depilated,
and prepared for surgery. The calvariae were exposed
using a midline scalp incision, and the skin was reflected
laterally to the supraorbital borders. Holes were then
made in the periosteum and bone using a fine dental bur
(0.4 mm) and packed with silver dental amalgam to serve
as radiopaque markers. The holes were placed in quadrants, 2 mm anterior and posterior to the coronal sutures,
and 2 mm lateral to the sagittal and interfrontal (IF)
sutures (Fig. 2). The animals all received postoperative IM
injections (2.5 mg/kg) of Baytril (Bayer Corp., Shawnee
Mission, KS) as a prophylaxis for infection.
The technique used to diagnosis rabbits with delayedonset synostosis was based on previously published criteria, and involved both the direct observation of suture
mobility and dysmorphology (Fig. 1) at 10 days of age and
the quantitative assessment of coronal suture growth
from serial radiographs (Mooney et al., 1994b, 1996a; Burrows et al., 1995; Losken et al., 1998, 1999). Cephalographs of the rabbits were taken at 10 and 25 days of age,
and rabbits in which coronal suture marker separation fell
below the 95% confidence interval of the mean for unaffected rabbits were classified as having delayed-onset synostosis. Rabbits with delayed-onset synostosis average
about 70 –75% of normal coronal suture growth, and usually fuse by 42 days of age (Fig. 1) (Mooney et al., 1994a;
Burrows et al., 1995; Losken et al., 1998, 1999).
At 25 days of age, after the initial diagnosis was reassessed and confirmed, the rabbits were randomly assigned
to one of the five groups. The rabbits were anesthetized,
their scalps were prepared for aseptic surgery, and the
calvariae were again exposed using a midline scalp incision as described above. An incision was then made in the
periosteum in the midline, and a small periosteal elevator
was used to create bilateral tunnels in the periosteum
superficial to the coronal sutures. The periosteal tunnels
were approximately 2 mm wide and 10 mm long, and
continued laterally along the coronal suture to the intersection of the coronal and squamosal sutures (pterion) on
both sides (Fig. 2).
In the sham control group rabbits, only the periosteal
tunnels were created and nothing was injected. The periosteal and skin incisions were then closed with 4-0 vicryl
suture. In the rabbits of the other four groups, 500 or
1,000 ng/suture of BSA or Tgf-␤3 were mixed with a collagen vehicle as described previously (Opperman et al.,
2002b), and injected through a 26G needle into the periosteal tunnels above the sutures. The vehicle was a highly
purified, slow-resorbing (⬎63 days in rabbit perisutural
tissues (Moursi et al., unpublished data)), bovine collagen
type I gel provided by NeuColl, Inc. (Campbell, CA). The
BSA and Tgf-␤3 were obtained from Sigma-Aldrich (St.
Louis, MO) and R&D Systems (Minneapolis, MN), respectively. The BSA and Tgf-␤3 were mixed under sterile
conditions with 100-␮l aliquots of the collagen gel to a
final concentration of 500 or 1,000 ng per gel aliquot in a
1-ml syringe. This volume ensured that the entire periosteal tunnel was filled with vehicle and protein. Following
injections, the periosteal and skin incisions were closed
with 4-0 vicryl suture.
Data Collection
Body weights and radiometry. Longitudinal body
weight and coronal suture growth data were obtained
from all rabbits at 10, 25, 42, and 84 days of age (at this
age, approximately 80 –90% of calvarial and brain growth
is completed in the rabbit) (Harel et al., 1972; Masoud et
al., 1986; Mooney et al., 1994b; Burrows et al., 1995).
Serial body weights were taken with a Tanita digital
scale (NLS Animal Health, Baltimore, MD). Serial lateral
and dorsoventral head radiographs were taken with the
rabbits tranquilized with an IM injection (0.40 ml/kg) of a
solution of 9l% Ketaset (ketamine hydrochloride, 100 mg/
ml; Aveco Co. Inc., Fort Dodge, IA) and 9% Rompun (xy-
SUTURE FUSION RESCUE USING Tgf-␤3
965
Fig. 2. A: Drawing of a rabbit skull showing amalgam marker placement, collagen injection site and harvesting area (hatched area), and
sagittal sectioning plane (dashed line) in the middle of a right coronal
suture. Although it was not drawn, the left coronal suture was sectioned
in the same plane. Histophotomicrographs (original magnification ⫽
25⫻) of a rabbit coronal suture in the sagittal plane showing the ecto-,
meso-, and endocortical landmarks used for measuring coronal suture
width (B) and the boundaries of the coronal suture that were traced
(white lines) to quantify the coronal suture area (C).
lazine, 20 mg/ml; Mobay Corp., Shawnee, KS). The heads
were immobilized in a specially designed cephalostat, and
a Philips Oralix 70 dental x-ray unit (Shelton, CT) was
used at an exposure of 50kV, 7mA, with a .17–.50-sec
exposure time, and a tube-to-cassette distance held constant at 152 cm (Mooney et al., 1994b; Burrows et al.,
1995; Losken et al., 1998, 1999). The radiographs were
viewed on a light box, and the amalgam markers at the
coronal suture were identified and traced on acetate tracing paper (Figs. 2 and 3). The tracings were then scanned
using a Hewlett-Packard ScanJet 5370C scanner and the
digital images were stored on a Gateway2000 PC. The
coronal suture markers were assigned Cartesian (x and y)
coordinates, and the distances between the markers were
measured using the Vistadent image analysis software
program. All measurements were taken blindly with regard to group identity. A random sample of 10% of the
radiographs were traced and measured twice. Intraobserver, repeated-measures reliability was calculated at
r ⫽ 0.975; P ⬍ 0.01, with a 3.45% standard error (S.E.) of
measurement.
formalin, demineralized in a formic acid solution (Calex II;
Fisher Scientific, Pittsburgh, PA), dehydrated in a series
of alcohol washes, and embedded in paraffin. The specimens were sectioned in the sagittal plane in the middle of
both the right and left coronal sutures (Fig. 2A) at a
thickness of 5–7 ␮. The middle of each coronal suture was
chosen for analysis because it is the focal point of synostosis in these rabbits (from this point, synostosis
progresses in both medial and lateral directions) (Mooney
et al., 1996b, 2002). Thus, this area is the most osteogenic
and should be most affected by cytokine manipulation. For
each suture, three sections were stained at 30-␮ intervals
with hematoxylin and erythrosin for conventional, qualitative bright-field light microscopy and histomorphometric analysis. This resulted in a sample of 300 sections for
histomorphometry (100 sutures ⫻ 3 sections/suture).
Histomorphometry of suture width and area for each
section was performed using a Leica MZ12 Stereo Zoom
microscope and Northern Eclipse (v 5.0) Image Analysis
Software (Empix Imaging, Inc., New York, NY). Digital
images of the specimens were captured using a Sony DKC5000, 3 CCD digital camera attached to the microscope
and stored on a Acer PC. Sutural width was taken at three
cortical levels (Fig. 2B) and defined as the greatest distance between the anterior and posterior edges of the
osteogenic fronts at the ecto- and endocortical surfaces
and in the middle of the cortical bone (mesocortical). The
Histomorphometry. At 84 days of age (56 days postoperatively), the rabbits were euthanized with an IV (40
mg/kg) injection of pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL), and the right and left sutures (n ⫽ 100) were harvested for histological examination. The specimens were fixed in 10% buffered neutral
966
CHONG ET AL.
suture widths, and suture area were calculated and compared among groups using a one-way analysis of variance
(ANOVA). Significant intergroup differences were assessed with the least-significant-difference multiple-comparisons test. All data were analyzed using SPSS 9.0 for
Windows. Differences were considered significant if P ⬍
0.05.
RESULTS
Fig. 3. Graphs showing mean (⫾S.E.) body weight (bottom) and
coronal suture marker separation (top) by group. No significant differences in somatic growth were found among the groups, but note the
significantly increased suture marker separation in the rabbits receiving
high-dose Tgf-␤3 (1,000 ng) compared to the other four groups.
anterior and posterior surfaces of the ecto-, meso-, and
endocortical levels of the frontal and parietal bones for
each section were identified on the stored digital images,
and the widths between them were measured (Fig. 2B)
using Northern Eclipse (v 5.0) Image Analysis Software
(Empix Imaging, Inc., New York, NY). The boundary of
the suture was also traced manually and the area was
calculated for each section (Fig. 2C). All measurements
were taken blindly with regard to group identity. A random sample of 10% of the images was traced and measured twice. Intraobserver, repeated-measures reliability
was calculated at r ⫽ 0.974; P ⬍ 0.001, with a 4.34% S.E.
of measurement for suture width and r ⫽ 0.970; P ⬍ 0.001,
and a 3.63% S.E. of measurement for suture area.
Statistical Analysis
Means and standard deviations for body weight and
coronal suture marker separation at each age, the three
All of the rabbits tolerated the surgical procedures very
well and no postoperative complications or deaths were
noted. Longitudinal body weight (Fig. 3) showed no significant group differences at 10 (F ⫽ 0.10; P ⬎ 0.05), 25 (F ⫽
0.10; P ⬎ 0.05), 42 (F ⫽ 1.08; P ⬎ 0.05), or 84 (F ⫽ 0.90;
P ⬎ 0.05) days of age, indicating that somatic growth was
unaffected by the surgery or cytokine treatment.
We assessed longitudinal coronal suture growth by analyzing coronal suture marker separation at various postoperative intervals. No significant (F ⫽ 1.87; P ⬎ 0.05)
differences in coronal suture marker separation were
noted among the five groups at 25 days of age (Fig. 3). In
contrast, rabbits with high-dose Tgf-␤3 (1,000 ng) had
significantly greater marker separation at 42 (F ⫽ 4.02;
P ⬍ 0.01) and 84 days of age (F ⫽ 3.83; P ⬍ 0.05) compared
to the other four groups. No significant differences were
noted among the remaining four groups at any age.
At 84 days of age (59 days posttreatment), the coronal
sutures were harvested for histological evaluation (Fig. 4).
A coronal suture from a wild-type rabbit was included for
comparison (Fig. 4A). The wild-type suture (outlined in
white) shows normal ventral and dorsal overlaps between
the parietal and frontal bones, and a consistent, homogeneous suture width at all three cortical locations (Fig. 4A).
In contrast, the coronal suture from the sham control
rabbit with delayed-onset synostosis was very narrow,
with a variable number of sutural bones and bony bridging in the endocortical region (Fig. 4B). The suture was
very heterogeneous, discontinuous, and vacuous. The osteogenic fronts were very dense and thickened, and in
some cases showed coronal ridging on the ectocortical
surface. Delayed-onset rabbit sutures that received BSA
(both groups) and a low dose of Tgf-␤3 (500 ng) showed
very similar morphologies to the sham control sutures,
although there was considerable phenotypic variability
(Fig. 4C–E). In contrast, delayed-onset rabbit sutures that
received a high dose of Tgf-␤3 (1,000 ng) showed very
patent and widened sutures, especially on the ectocortical
surface at the site of the Tgf-␤3 injections (Fig. 4F). The
sutures were more fibrotic and homogeneous in consistency, especially in the ecto- and mesocortical regions that
were closer to the injection sites.
Histomorphometry of the sutures supported the qualitative results (Fig. 5). One-way ANOVA revealed significant group differences in suture width for all three cortical
regions. For the ectocortical region (F ⫽ 17.62; P ⬍ 0.001),
multiple-comparisons tests revealed that rabbit sutures
treated with a high dose of Tgf-␤3 (1,000 ng) had significantly (P ⬍ 0.05) wider sutures than the other four groups
(Fig. 5). No significant differences (P ⬎ 0.05) in ectocortical width were noted among the other four groups. For the
mesocortical region (F ⫽ 5.23;P ⬍ 0.001), multiple-comparisons tests revealed that rabbit sutures treated with a
high dose of Tgf-␤3 (1,000 ng) had significantly (P ⬍ 0.05)
wider sutures than the other four groups. The sham controls and the high-dose BSA (1,000 ng) groups had signif-
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SUTURE FUSION RESCUE USING Tgf-␤3
Fig. 4. Histophotomicrographs (original magnification ⫽ 25⫻) of
coronal sutures from 84-day-old rabbits, showing the osteogenic fronts
of the parietal (P) and frontal (F) bones and the coronal suture (arrows) for
the various groups. The suture mesenchyme is outlined in white for
comparison. Note the very narrow coronal suture with bony bridging
(arrow) in the sham control group (B) and the widened and patent
coronal suture in the high-dose Tgf-␤3 suture (F) compared to the other
groups.
icantly (P ⬍ 0.05) wider sutures than the groups treated
with a low-dose of BSA and Tgf-␤3 (500 ng). Similar findings were noted for the ectocortical region (F ⫽ 3.82; P ⬍
0.01). Multiple-comparisons tests revealed that rabbit sutures treated with a high dose of Tgf-␤3 (1,000 ng) had
significantly (P ⬍ 0.05) wider sutures than the other four
groups. The sham controls and the high-dose BSA (1,000
ng) groups had significantly (P ⬍ 0.05) wider sutures than
the groups treated with a low dose of BSA and Tgf-␤3 (500
ng) (Fig. 5).
Significant group differences were also noted for suture
area (F ⫽ 4.21; P ⬍ 0.01). Multiple-comparisons tests
revealed that rabbit sutures treated with a high dose of
Tgf-␤3 (1,000 ng) had significantly (P ⬍ 0.05) greater
suture area than the other four groups (Fig. 5). No significant differences (P ⬎ 0.05) in suture area were noted
among the other four groups.
DISCUSSION
The Tgf-␤s are a superfamily of growth factors that
comprises over two dozen related polypeptides, including
the Bmps (Centrella et al., 1994; Massague et al., 1994;
Cohen, 2000c). The Tgf-␤s play an essential role in many
biological processes, including collagen synthesis, bone
regeneration, suture patency, and eventual suture fusion
(Centrella et al., 1994; Massague et al., 1994; Opperman
et al., 1997, 1999, 2000, 2002a,b; Roth et al., 1997a,b;
Cohen, 2000c; Opperman and Ogle, 2002). The manipulation of these growth factors may also have clinical applications in the treatment of craniosynostosis (Roth et al.,
1997b; Opperman et al., 1999, 2000, 2002b; Chong et al.,
2001, 2002; Mooney et al., 2002a; Opperman and Ogle,
2002). The current results support this premise and demonstrate that a course of treatment with Tgf-␤3 can rescue
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CHONG ET AL.
Fig. 5. Graphs showing mean (⫾S.E.) coronal suture width (top) and
coronal suture area (bottom) by group. Note the significantly increased
coronal suture width and coronal suture area in the rabbits receiving
high-dose Tgf-␤3 (1,000 ng) compared to the other four groups.
fusing coronal sutures in a rabbit model of delayed-onset
craniosynostosis.
It has been suggested that the delayed-onset condition
may be a variable phenotypic expression of a familial
craniosynostotic condition, and may represent part of a
synostotic continuum (Reddy et al., 1990; Hoffman and
Reddy, 1991; Cohen et al., 1993; Mooney et al., 1994a,b,
1996a, 2002b; Burrows et al., 1995; Losken et al., 1998,
1999). It is thought that this continuum ranges from 1)
normal growing sutures in affected genotypes on one end
to 2) delayed-onset and single-suture synostosis in the
middle of the continuum, and then to 3) early-onset and
multiple-suture synostosis at the other extreme. Although
the pathogenesis of delayed-onset synostosis is unknown,
dense collagen bundles, small bony bridges, and an in-
creased number of sutural bones have all been observed in
the coronal sutures of rabbits with delayed-onset synostosis (Mooney et al., 1994a,b, 1996a; Burrows et al., 1995,
1997; Losken et al., 1998, 1999). These bundles, bridges,
and supernumerary bones probably immobilize the suture
and result in neurocranial growth restrictions and altered
neurocapsular growth vectors, as observed in both the
rabbit model (Mooney et al., 1994b; Burrows et al., 1995;
Losken et al., 1998) and clinically (Reddy et al., 1990;
Hoffman and Reddy, 1991; Cohen et al., 1993; Cohen and
MacLean, 2000). The partially immobilized coronal sutures in rabbits from our colony usually synostose at
42– 84 days of age (normal suture fusion in rabbits occurs
at about 3– 4 years of age (Persson et al., 1978)), as observed histologically in sham control rabbits in the
present study and in previous investigations (Mooney et
al., 1994b, 2002b; Burrows et al., 1995; Losken et al., 1998,
1999). Coronal suture synostosis was prevented by the use
of Tgf-␤3 in these rabbits.
Although the specific causal mechanism of Tgf-␤3-mediated suture fusion rescue is still unclear (Opperman et
al., 2002b), it is thought that Tgf-␤3 regulates suture
patency by regulating suture cell proliferation and apoptosis through interactions with other Tgf-␤ isoforms and
their receptors (Opperman and Ogle, 2002; Opperman et
al., 2002b). Opperman and colleagues recently showed
that small doses of Tgf-␤3 (3–30 ng) were associated with
a reduced number of suture fibroblasts that were immunoreactive for T␤r-I in rat IF sutures both in vivo (Opperman et al., 2002b) and in vitro (Opperman et al., 2002a).
Since Tgf-␤2 and Tgf-␤3 share the same set of transmembrane receptors and the same intracellular Smads (Centrella et al., 1996), downregulation of T␤r-I expression by
Tgf-␤3 may limit receptor access, thereby reducing the
mitogenic and osteogenic effects of Tgf-␤2. This may explain, in part, the antagonistic effects of these two isoforms in regulating suture morphogenesis and fusion (Opperman et al., 2000; Opperman and Ogle, 2002). While
Tgf-␤s can exhibit distinct effects with the use of the same
receptors, it also appears that different Tgf-␤ receptors
can have distinct activities in response to different growth
factor binding agents (Boyer and Runyan, 2001). Thus, it
is likely that one Tgf-␤ can regulate the activity of another, either by regulating different signaling pathways or
by regulating access to the Tgf-␤ receptors (Opperman et
al., 2002a,b). Rice et al. (1999) demonstrated in vivo that
osteoclast apoptosis is necessary for normal suture formation and the maintenance of suture patency. Opperman et
al. (2000) reported decreased cell proliferation and apoptosis in rat IF sutures with Tgf-␤3 exposure. These findings
suggest that Tgf-␤3 may also help maintain suture patency by controlling the osteogenic rate and maintaining
the balance between normal bone formation and resorption in the osteogenic fronts (Opperman et al., 2000, Opperman et al., 2002a,b).
Tgf-␤3 is also known to be a biphasic regulator of osteoblastic activity: lower concentrations of Tgf-␤3 stimulate
cell proliferation, and higher concentrations of Tgf-␤3 inhibit cell proliferation and stimulate extracellular matrix
production and osteoid formation (ten Dijke et al., 1990).
Opperman et al. (2002b) demonstrated a dose-dependent
effect of Tgf-␤3 in a study in which a single dose of 3 ng of
Tgf-␤3 rescued normal IF sutures from fusing in 9-day-old
rat pups, whereas a 10-fold higher dose (30 ng) did not. In
the present study, 1,000 ng of Tgf-␤3 rescued fusing coro-
SUTURE FUSION RESCUE USING Tgf-␤3
nal sutures, whereas 500 ng of Tgf-␤3 failed to rescue
sutures from bridging and fusing. These differences may
be explained by a number of factors. The data in the
Opperman et al. (2002b) study were collected from normal
rat sutures undergoing normal suture fusion. In contrast,
in the present study, the data were obtained from pathological rabbit sutures. While the specific genetic mutation
responsible for craniosynostosis in these rabbits has not
yet been identified, it is possible that differences in Tgf-␤
receptor morphology or expression, and/or intracellular
signaling pathways between normal and pathological
groups may affect Tgf-␤3 activity. Furthermore, the initial
Tgf-␤3 dosages used in the present study were based on in
vitro data collected on rat osteoblastic activity (Moursi et
al., unpublished data). It is possible that due to speciesspecific differences, rodents are more sensitive to Tgf-␤3
than rabbits, as indicated by the bioactive dosage levels
(30 ng) used by Opperman et al. (2002a) compared to those
used in the present study (1,000 ng). Finally, the actual
amounts of Tgf-␤3 released from the collagen gel were not
quantified in the perisutural tissues, and the sutures in
the Tgf-␤3 groups may have received smaller amounts
than anticipated. However, studies using this collagen gel
vehicle have shown that Tgf-␤2 mixed with the collagen
gel in culture retained a level of activity similar to that of
Tgf-␤2 alone (Schroeder-Tefft et al., 1997; Bentz et al.,
1998). The collagen lasted for at least 63 days in rabbit
perisutural tissue, as observed by the use of biotinylated
collagen gel (Moursi et al., unpublished data), and it
showed no evidence of a localized inflammatory or generalized immune response following gel implantation
(Chong et al., 2001, 2002; Mooney et al., 2002a; Opperman
et al., 2002b) (Moursi et al., unpublished data). However,
biokinetic and degradation studies still need to be performed in this model to quantify the actual bioactive dosages.
The results from the present study demonstrate that
Tgf-␤3 administration rescued fusing coronal sutures and
improved coronal suture growth (as evidenced by longitudinal amalgam marker separation) in a rabbit craniosynostosis model. The significant increase in coronal suture
marker separation in rabbits treated with a high dose of
Tgf-␤3 compared to the other groups may be a primary
result of hyperplasia of the fibrous suture ligament “pushing” the amalgam markers apart. Alternatively, it may be
a secondary effect of compensatory growth stretch from
neural capsule expansion once the sutural tethering from
the bony bridges was relaxed from apoptosis of the osteoblasts in the osteogenic fronts. Additional data are needed
to determine which growth mechanism may be operating,
and researchers should use caution when extrapolating
these data to the clinical setting. A myriad of genetic and
epigenetic etiologies that produce craniosynostosis have
been identified (Cohen, 1989, 2000a; Jabs, 2002), and this
rabbit strain only models a small percentage of craniosynostotic cases (Mooney et al., 1994a, 1996a, 2002; Cohen,
2000b). However, the highly conserved Tgf-␤3 signaling
pathway for suture maintenance is probably active in the
majority of these etiologies (Centrella et al., 1994; Massague et al., 1994; Cohen, 2000c; Opperman and Ogle,
2002), and can be manipulated. Variations in neurocapsular growth patterns and timing have also been observed in
rabbits and humans. Approximately 90% of brain growth
in rabbits is completed by 35 days of age (Harel et al.,
1972; Kier, 1976; Alberius and Selvik, 1985; Masoud et al.,
969
1986; Cooper et al., 1999), as compared to about 4 – 6 years
of age in humans (Enlow and McNamara, 1973; Kier,
1976; Enlow, 1990). Thus, in humans a much greater
length of time would be needed for sutural exposure to
Tgf-␤3 to prevent premature suture fusion. This could be
accomplished by using repeated dosing, alternative longerterm degrading delivery vehicles or systems, or gene therapy. It is also difficult to diagnose delayed-onset synostosis in humans. In the clinical literature, delayed-onset
synostosis has been fortuitously identified in a small sample of children ranging from 2 to 10 years of age (Reddy et
al., 1990; Hoffman and Reddy, 1991; Cohen et al., 1993;
Cohen and MacLean, 2000). These diagnoses were based,
in part, on secondary clinical findings of progressively
elevated intracranial pressure and papilledema, developmental delays, and mild craniofacial malformations
(Reddy et al., 1990; Hoffman and Reddy, 1991; Cohen et
al., 1993; Cohen and MacLean, 2000), all of which were
observed following postgestational suture fusion. To increase the effectiveness of “rescue” therapy, improvements must be made in the early diagnosis of this condition. However, even with the described limitations, the
rabbits in the current study appear to be an appropriate
model of human familial, nonsyndromic coronal suture
synostosis (Mooney et al., 1994a, 1996a, 2002; Cohen,
200b), and should prove to be useful for testing cytokine
therapies for the treatment of craniosynostosis.
ACKNOWLEDGMENTS
The authors thank the anonymous reviewers of The
Anatomical Record for their thoughtful comments and
helpful criticisms of this manuscript. NeuColl, Inc. supplied the collagen vehicle.
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