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.
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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:
[email protected] 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
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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 Tr-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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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-
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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
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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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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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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 Tr-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 Tr-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.,
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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.
LITERATURE CITED Alberius P, Selvik G. 1985. Volumetric changes in the developing rabbit calvarium. Anat Rec 213:207–214. Arnaud E, Renier D, Marchac D. 1995. Prognosis for mental function in scaphocephaly. J Neurosurg 83:476 – 479. Babler WJ, Persing JA. 1982. Experimental alteration of cranial suture growth: effects on the neurocranium, basicranium, and midface. In: Sarnat BG, editor. Factors and mechanisms influencing bone growth. New York: Alan R. Liss, Inc. p 333–345. Babler WJ. 1989. Relationship of altered cranial suture growth to cranial base and midface. In: Persing JA, Edgerton MT, Jane JA, editors. Scientific foundations and surgical treatment of craniosynostosis. Baltimore: Williams and Wilkins. p 87–95. Bentz H, Schroeder JA, Estridge TD. 1998. Improved local delivery of TGF-2 by binding to injectable fibrillar collagen via difunctional polyethylene glycol. J Biomed Mater Res 394:539 –548. Boyer AS, Runyan RB. 2001. TGF- type III and TGF- type II receptors have distinct activities during epithelial-mesenchymal cell transformation in the embryonic heart. Dev Dyn 2214:454 – 459. Burdi AR, Kusnetz AB, Venes JL, Gebarski SS. 1986. The natural history of the cranial coronal ring articulations: implications in understanding the pathogenesis of the Crouzon craniosynostotic defects. Cleft Palate J 23:28 –39. Burrows AM, Mooney MP, Smith TD, Losken HW, Siegel MI. 1995. Growth of the cranial vault changes in rabbits with congenital coronal suture synostosis. Cleft Palate Craniofac J 32:235–246. Burrows AM, Caruso KA, Mooney MP, Smith TD, Losken HW, Siegel MI. 1997. Sutural bone frequency in synostotic rabbit crania. Am J Phys Anthropol 102:555–563. Camfield PR, Camfield CS, Cohen Jr MM. 2000. Neurological aspects of craniosynostosis. In: Cohen MM, MacLean RE, editors. Craniosynostosis: diagnosis, evaluation, and management. 2nd ed. New York: Oxford University Press. p 177–184.
970
CHONG ET AL.
Centrella M, Horowitz MC, Wozney JM, McCarthy TL. 1994. Transforming growth factor- gene family members and bone. Endocrinol Rev 15:27–37. Centrella M, Ji C, Casinghino S, McCarthy TL. 1996. Rapid flux in transforming growth factor-beta receptors on bone cells. J Biol Chem 27131:18616 –18622. Chong SL, Mitchell R, Moursi A, Winnard P, Losken HW, Ozerdem O, Keeler K, Opperman L, Siegel MI, Mooney MP. 2001. Rescue of coronal suture synostosis with TGF-3 in craniosynostotic rabbits. J Dent Res 80:89. Chong SL, Mitchell R, Moursi A, Winndard P, Losken HW, Azari K, Change T, Bradley J, Opperman LA, Siegel MI, Mooney MP. 2002. The effects of TGF-3 on coronal suture fusion in craniosynostotic rabbits. J Dent Res 81:A– 462. Cohen MM. 1979. Craniosynostosis and syndromes with craniosynostosis: incidence, genetics, penetrance, variability, and new syndrome updating. Birth Defects 15(5B):13– 63. Cohen MM. 1989. The etiology of craniosynostosis. In: Persing JA, Edgerton MT, Jane JA, editors. Scientific foundations and surgical treatment of craniosynostosis. Baltimore: Williams & Wilkins. p 9 –20. Cohen Jr MM, Kreiborg S. 1992. Birth prevalence studies of the Crouzon syndrome: comparison of direct and indirect methods. Clin Genet 41:12–15. Cohen Jr MM. 2000a. Fibroblast growth factor receptor mutations. In: Cohen MM, MacLean RE, editors. Craniosynostosis: diagnosis, evaluation, and management. 2nd ed. New York: Oxford University Press. p 77–94. Cohen Jr MM. 2000b. Sutural pathology. In: Cohen Jr MM, MacLean RE, editors. Craniosynostosis: diagnosis, evaluation, and management. New York: Oxford University Press. p 95–99. Cohen Jr MM. 2000c. TGF and suture biology. In: Cohen Jr MM, MacLean RE, editors. Craniosynostosis: diagnosis, evaluation, and management. New York: Oxford University Press. p 69 –76. Cohen Jr MM, MacLean RE. 2000. Anatomic, genetic, nosologic, diagnostic, and psychosocial considerations. In: Cohen Jr MM, MacLean RE, editors. Craniosynostosis: diagnosis, evaluation, and management. New York: Oxford University Press. p 119 –146. Cohen SR, Dauser RC, Gorski JL. 1993. Insidious onset of familial craniosynostosis. Cleft Palate Craniofac J 30:401– 405. Cooper GM, Mooney MP, Burrows AM, Smith TD, Dechant JJ, Losken HW, Marsh JL, Siegel MI. 1999. Brain growth rates in craniosynostotic rabbits. Cleft Palate Craniofac J 36:314 –321. Dufresne C, Richtsmeier JT. 1995. The interaction of craniofacial dysmorphology, growth, and the prediction of surgical outcome. J Craniofac Surg 4:270 –281. Enlow DH, McNamara J. 1973. The neurocranial basis for facial form and pattern. Angle Orthod 43:256 –262. Enlow DH. 1990. Handbook of facial growth. Philadelphia: Saunders. p 288. Fatah MFT, Ermis I, Poole MD, Shun-Shin GA. 1992. Prevention of cranial reossification after surgical craniectomy. J Craniofac Surg 3:170 –172. Fellows-Mayle W, Mooney MP, Dechant J, Losken HW, Cooper GM, Burrows AM, Smith TD, Pollack I, Siegel MI. 2000. Longitudinal intracranial pressure changes in craniosynostotic rabbits. Cleft Palate Craniofac J 37:370 –378. Gault DT, Renier D, Marchac D, Jones BM. 1992. Intracranial pressure and intracranial volume in children with craniosynostosis. Plast Reconstr Surg 90:377–381. Harel S, Watanabe K, Linke I, Schain RJ. 1972. Growth and development of the rabbit brain. Biol Neonate 21:381–399. Hoffman HJ, Reddy KKV. 1991. Progressive cranial suture stenosis in craniosynostosis. Neurosurg Clin N Am 2:555–564. Hoyte DAN. 1989. The role of the cranial base in normal and abnormal skull development. In: Persing JA, Edgerton MT, Jane JA, editors. Scientific foundations and surgical treatment of craniosynostosis. Baltimore: Williams & Wilkins. p 58 –76. Hudgins RJ, Cohen SR, Burstein FD, Boydston WR. 1998. Multiple suture synostosis and increased intracranial pressure following repair of single suture, nonsyndromic craniosynostosis. Cleft Palate Craniofac J 35:167–172.
Jabs EW. 2002. The genetics of craniosynostosis. In: Mooney MP, Siegel MI, editors. Understanding craniofacial anomalies: the etiopathogenesis of craniosynostosis and facial clefting. New York: John W. Wiley and Sons. p 125–146. Kapp-Simmonds KA, Figueroa A, Jocher CA, Schafer M. 1993. Longitudinal assessment of mental development in infants with nonsyndromic craniosynostosis with and without cranial release and reconstruction. Plast Reconstr Surg 92:831– 841. Kier EL. 1976. Phylogenetic and ontogenetic changes of the brain relevant to the evolution of the skull. In: Bosma J, editor. Development of the basicranium. Bethesda, MD: DHEW (NIH). p 468. Lin KY, Nolen AA, Gampper TJ, Jane JA, Opperman LA, Ogle RC. 1997. Elevated levels of transforming growth factors beta 2 and beta 3 in lambdoid sutures from children with persistent plagiocephaly. Cleft Palate Craniofac J 34:331–337. Losken HW, Mooney MP, Zoldos J, Tschakaloff A, Burrows AM, Smith TD, Cooper GM, Kapucu MR, Siegel MI. 1998. Internal calvarial bone distraction in rabbits with delayed-onset coronal suture synostosis. Plastic Reconstr Surg 102:1109 –1119. Losken HW, Mooney MP, Zoldos J, Tschakaloff A, Burrows AM, Smith TD, Cano G, Arnott R, Sherwood C, Dechant J, Cooper GM, Kapucu MR, Siegel MI. 1999. Coronal suture response to distraction osteogenesis in rabbits with delayed-onset craniosynostosis. J Craniofac Surg 10:27–37. Marchac D, Renier D. 1982. Craniofacial surgery for craniosynostosis. Boston: Little Brown & Co. p 114 –123. Marsh JL, Vannier MW. 1985. Comprehensive care for craniofacial deformities. St. Louis: C.V. Mosby. p 72–79. Marsh JL, Vannier MW. 1986. Cranial base changes following surgical treatment of craniosynostosis. Cleft Palate J 23(suppl):9 –18. Masoud I, Shapiro, Moses A. 1986. Longitudinal roentgen cephalometric study of the growth of the New Zealand White rabbit: cumulative and biweekly incremental growth rates for skull and mandible. J Craniofac Gen Dev Biol 6:259 –287. Massague J, Attisano L, Wrana JL. 1994. The TGF- family and its composite receptors. Trends Cell Biol 4:172–178. Miller MT. 2000. Ocular findings in craniosynostosis. In: Cohen MM, MacLean RE, editors. Craniosynostosis: diagnosis, evaluation, and management. 2nd ed. New York: Oxford University Press. p 184 – 196. Mooney MP, Losken HW, Siegel MI, Lalikos JF, Losken A, Smith TD, Burrows A. 1994a. Development of a strain of rabbits with congenital simple, nonsyndromic coronal suture synostosis. Part 1. Breeding demographics, inheritance pattern, and craniofacial anomalies. Cleft Palate Craniofac J 31:1–7. Mooney MP, Losken HW, Siegel MI, Lalikos JF, Losken A, Burrows A, Smith TD. 1994b. Development of a strain of rabbits with congenital simple, nonsyndromic coronal suture synostosis. Part 2. Somatic and craniofacial growth patterns. Cleft Palate Craniofac J 31:8 –14. Mooney MP, Aston CE, Siegel MI, Losken HW, Smith TD, Burrows AM, Wenger SL, Caruso K, Siegel B, Ferrell RE. 1996a. Craniosynostosis with autosomal dominant transmission in New Zealand White rabbits. J Craniofac Gen Dev Biol 16:52– 63. Mooney MP, Smith TD, Burrows AM, Langdon HL, Stone CE, Losken HW, Caruso K, Siegel MI. 1996b. Coronal suture pathology and synostotic progression in rabbits with congenital craniosynostosis. Cleft Palate Craniofac J 33:369 –378. Mooney MP, Burrows AM, Wigginton W, Singhal VK, Losken HW, Smith TD, Dechant J, Towbin A, Cooper G, Towbin R, Siegel MI. 1998a. Intracranial volume in craniosynostotic rabbits. J Craniofac Surg 9:234 –239. Mooney MP, Siegel MI, Burrows AM, Smith TD, Losken HW, Dechant J, Cooper G, Fellow-Mayle W, Kapucu MR, Kapucu LO. 1998b. A rabbit model of human familial, nonsyndromic, unicoronal suture synostosis: part 2. Intracranial contents, intracranial volume, and intracranial pressure. Childs Nerv Syst 14:247–255. Mooney MP, Siegel MI, Burrows AM, Smith TD, Losken HW, Dechant J, Cooper G, Kapucu MR. 1998c. A rabbit model of human familial, nonsyndromic, unicoronal suture synostosis: part 1. Synostotic onset, pathology, and sutural growth patterns. Childs Nerv Syst 14: 236 –246.
SUTURE FUSION RESCUE USING Tgf-3 Mooney MP, Fellows-Mayle W, Dechant J, Losken HW, Burrows AM, Smith TD, Cooper GM, Pollack I, Siegel MI. 1999. Increases in intracranial pressure following coronal suturectomy in rabbits with craniosynostosis. J Craniofac Surg 10:104 –111. Mooney MP, Losken HW, Moursi A, Mitchell R, Winnard P, Ozerdem O, Bradley J, Azari K, Acarturk O, Chang T, Opperman LA, Stelnicki EJ, Siegel MI. 2002a. Inhibition of postoperative resynostosis with anti-TGF-2 antibody. J Dent Res 81:A–221. Mooney MP, Siegel MI, Opperman LA. 2002b. Animal models of craniosynostosis: Experimental, congenital, and transgenic. In: Mooney MP, Siegel MI, editors. Understanding craniofacial anomalies: the etiopathogenesis of craniosynostosis and facial clefting. New York: John W. Wiley and Sons. p 209 –250. Most D, Levine JP, Chang J, Sung J, McCarthy JG, Schendel SA, Longaker MT. 1998. Studies in cranial suture biology: upregulation of transforming growth factor-1 and basic fibroblast growth factor mRNA correlates with posterior frontal cranial suture fusion in the rat. Plastic Reconstr Surg 101:1431–1440. Opperman LA, Nolen AA, Ogle RC. 1997. TGF-1, TGF-2, and TGF-3 exhibit distinct patterns of expression during cranial suture formation and obliteration in vivo and in vitro. J Bone Miner Res 12:301–310. Opperman LA, Chhabra A, Cho RW, Ogle RC. 1999. Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-3 activity and prevented by removal of TFG-2 activity from fetal rat calvaria in vitro. J Craniofac Genet Dev Biol 19:164 – 173. Opperman LA, Adab K, Gakunga PT. 2000. Transforming growth factor-2 and TGF-3 regulate fetal rat cranial suture morphogenesis by regulating rates of cell proliferation and apoptosis. Dev Dyn 219:237–247. Opperman LA, Ogle RC. 2002. Molecular studies of craniosynostosis: factors affecting cranial suture morphogenesis and patency. In: Mooney MP, Siegel MI, editors. Understanding craniofacial anomalies: the etiopathogenesis of craniosynostosis and facial clefting. New York: John W. Wiley and Sons. p 497–518. Opperman LA, Galanis V, Williams AR, Adab K. 2002a. Transforming growth factor-beta 3 (TGF-3) down-regulates TGF- receptor I (Tr-I) during rescue of cranial sutures from osseous obliteration. Orthodont Craniofac Res 5:5–16. Opperman LA, Moursi AM, Sayne JR, Wintergerst AM. 2002b. Transforming growth factor-beta 3 (TGF-3) in a collagen gel delays fusion of the rat posterior interfrontal suture in vivo. Anat Rec 267:120 –130. Ousterhout DK, Vargervik K. 1987. Aesthetic improvement resulting from craniofacial surgery in craniosynostosis syndromes. J Craniomaxillofac Surg 15:189 –197. Persing JA, Jane JA, Edgerton MA. 1989. Surgical treatment of craniosynostosis. In: Persing JA, Edgerton MT, Jane JA, editors. Scientific foundations and surgical treatment of craniosynostosis. Baltimore: Williams and Wilkins. p 87–95.
971
Persing JA, Jane JA. 2000. Neurosurgical treatment of craniosynostosis. In: Cohen MM, MacLean RE, editor. Craniosynostosis: diagnosis, evaluation, and management. 2nd ed. New York: Oxford University Press. p 209 –227. Persson M, Magnusson BC, Thilander B. 1978. Sutural closure in rabbit and man: a morphological and histochemical study. J Anat 125: 313–321. Poisson E, Mooney MP, Koepsel R, Cooper GM, Opperman LA, Sciote JJ. 1999. TGF- isoform expression in the perisutural tissues of craniosynostotic rabbits. J Dent Res 78:363. Posnick JC. 2000. Craniofacial and maxillofacial surgery in children and young adults. Vols. I and II. Philadelphia: W.B. Saunders. Reddy K, Hoffman HJ, Armstrong D. 1990. Delayed and progressive multiple suture craniosynostosis. Neurosurgery 26:442– 448. Renier D. 1989. Intracranial pressure in craniosynostosis: pre- and postoperative recordings— correlation with functional results. In: Persing JA, Edgerton MT, Jane JA, editors. Scientific foundations and surgical treatment of craniosynostosis. Baltimore: Williams & Wilkins. p 263–269. Rice D, Kim H, Thesleff I. 1999. Apoptosis in murine calvarial bone and suture development. Eur J Oral Sci 107:265–275. Roth DA, Gold LI, Han VKM, McCarthy JG, Sung JJ, Wisoff JH, Longaker MT. 1997a. Immunolocalization of transforming growth factor 1, 2, and 3 and insulin-like growth factor I in premature cranial suture fusion. Plastic Reconstr Surg 99:300 –309. Roth DA, Longaker MT, McCarthy JG, Rosen DM, McMullen HF, Levine JP, Sung J, Gold LI. 1997b. Studies in cranial suture biology: Part I. increased immunoreactivity for TGF  isoforms (1, 2, and 3) during rat cranial suture fusion. J Bone Miner Res 12:311–321. Schroeder-Tefft JA, Bentz H, Estridge TD. 1997. Collagen and heparin matrices for growth factor delivery. J Control Release 49:291– 298. Singhal VK, Mooney MP, Burrows AM, Wigginton W, Losken HW, Smith TD, Towbin R, Siegel MI. 1997. Age related changes in intracranial volume in craniosynostotic rabbits using 3-D CT scans. Plast Reconstr Surg 100:1121–1128. Smith TD, Mooney MP, Burrows AM, Losken HW, Siegel MI. 1996. Postnatal changes in the cranial base in rabbits with congenital coronal suture synostosis. J Craniofac Genet Dev Biol 16:107–117. ten Dijke P, Iwata KK, Goddard C, Pieler C, Canalis E, McCarthy TL, Centrella M. 1990. Recombinant transforming growth factor type 3: biological activities and receptor-binding properties in isolated bone cells. Mol Cell Biol 10:4473– 4479. Turvey TA, Vig KWL, Fonseca RL. 1996. Facial clefts and craniosynostosis: principles and management. Philadelphia: W.B. Saunders. p 596 – 685. Williams KJ, Cohen SR, Burstein FD, Hudgins R, Boydston W, Simms C. 1997. A longitudinal, statistical study of reoperation rates in craniosynostosis. Plast Reconstr Surg 100:305–310.
