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JOURNAL OF BONE AND MINERAL RESEARCH Volume 22, Number 7, 2007 Published online on April 16, 2007; doi: 10.1359/JBMR.070410 © 2007 American Society for Bone and Mineral Research

Noggin Inhibits Postoperative Resynostosis in Craniosynostotic Rabbits Gregory M Cooper,1 Chris Curry,2 Timothy E Barbano,3 Anne M Burrows,3,4 Lisa Vecchione,5,6 John F Caccamese,7 Craig S Norbutt,8 Bernard J Costello,8 Joseph E Losee,1,6 Amr M Moursi,9 Johnny Huard,10 and Mark P Mooney6,11

ABSTRACT: Inhibition of bone formation after surgery to correct craniosynostosis would alleviate the need for secondary surgeries and decrease morbidity and mortality. This study used a single dose of Noggin protein to prevent resynostosis and improve postoperative outcomes in a rabbit model of craniosynostosis. Introduction: Craniosynostosis is defined as the premature fusion of one or more of the cranial sutures, which causes secondary deformations of the cranial vault, cranial base, and brain. Current surgical intervention involves extirpation of the fused suture to allow unrestricted brain growth. However, resynostosis of the extirpated regions often occurs. Several bone morphogenetic proteins (BMPs), well-described inducers of ossification, are involved in bone healing. This study tested the hypothesis that a postoperative treatment with Noggin, an extracellular BMP inhibitor, can inhibit resynostosis in a rabbit model of human familial nonsyndromic craniosynostosis. Materials and Methods: Thirty-one New Zealand white rabbits with bilateral coronal suture synostosis were divided into three groups: (1) suturectomy controls (n ⳱ 13); (2) suturectomy with BSA in a slow-resorbing collagen vehicle, (n ⳱ 8); and (3) suturectomy with Noggin in a slow-resorbing collagen vehicle (n ⳱ 10). At 10 days of age, a 3 × 15-mm coronal suturectomy was performed. The sites in groups 2 and 3 were immediately filled with BSA-loaded gel or Noggin-loaded gel, respectively. Serial 3D-CT scan reconstructions of the defects and standard radiographs were obtained at 10, 25, 42, and 84 days of age, and the sutures were harvested for histological analysis. Results: Radiographic analysis revealed that Noggin-treated animals had significantly greater coronal suture marker separation by 25 days and significantly greater craniofacial length at 84 days of age compared with controls. 3D-CT analysis revealed that Noggin treatment led to significantly greater defect areas through 84 days and to increased intracranial volumes at 84 days of age compared with other groups. Histological analysis supported CT data, showing that the untreated and BSA-treated groups had significant healing of the suturectomy site, whereas the Noggin-treated group had incomplete wound healing. Conclusions: These data support our hypothesis that inhibition of BMP activity using Noggin may prevent postoperative resynostosis in this rabbit model. These findings also suggest that Noggin therapy may have potential clinical use to prevent postoperative resynostosis in infants with craniosynostosis. J Bone Miner Res 2007;22:1046–1054. Published online on April 16, 2007; doi: 10.1359/JBMR.070410 Key words: craniosynostosis, rabbit model, postoperative resynostosis, Noggin, bone healing

INTRODUCTION

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RANIOSYNOSTOSIS IS THE term given to the premature fusion of one or more of the cranial sutures and occurs with an estimated birth prevalence of 300–500 per 1,000,000 live births.(1) Primary craniosynostosis causes secondary de-

The authors state that they have no conflicts of interest.

formities in the cranial vault, cranial base, and in the midface(2,3) that can cause increased intracranial pressure(4,5) and altered intracranial volume(5–8) that can lead to blindness, cognitive deficiencies, and mental retardation if left uncorrected.(4,9) Current surgical management involves the extirpation of the fused suture along with extensive cranial vault reshaping.(10–14) Although current surgical strategies often allow for normal brain growth and development, re-establish normal in-

1 Department of Surgery, Division of Pediatric Plastic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 2Department of Oral Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 3School of Dental Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 4Department of Anthropology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 5Department of Physical Therapy, Rangos School of Health Sciences, Duquesne University, Pittsburgh, Pennsylvania, USA; 6Pittsburgh CleftCraniofacial Center, Pittsburgh, Pennsylvania, USA; 7Department of Oral and Maxillofacial Surgery, University of Maryland, Baltimore, Maryland, USA; 8Department of Oral and Maxillofacial Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 9Department of Pediatric Dentistry, College of Dentistry, New York University, New York, New York, USA; 10Departments of Orthopaedic Surgery, Molecular Genetics and Biochemistry, and Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA; 11Department of Orthodontics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

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FIG. 1. Animal model of craniosynostosis and surgical intervention. (A) Photograph showing the skulls of a normal, wildtype rabbit (left) compared with a rabbit with complete bilateral coronal suture synostosis (right). Both skulls were from 84-day-old rabbits. Note the lack of a coronal suture in the synostosed rabbit (arrow). (B) Intraoperative photograph showing the defect resultant from the suturectomy and the initial placement of the collagen gel. (C) Intraoperative photograph showing the final placement of the injectable collagen gel that was either mixed with BSA or Noggin. Note: the injectable nature of the gel allowed for precise placement of the treatment within the defect site.

tracranial volume and pressure, and correct cosmetic deformities, the suturectomy site frequently reossifies very rapidly after surgery (in 30–100% of reported cases and as early as 6 mo after surgery). Reossification of the suturectomy site leads to the reapproximation of the osteotomy margins and the refusion of the extirpated suture, termed “resynostosis.”(14–17) Such resynostosis can lead to a secondary increase in intracranial pressure and further restrict craniofacial growth,(18–20) necessitating additional surgical procedures(21) and increasing the risk of patient morbidity and mortality. The molecular mechanisms underlying resynostosis are poorly understood, although we believe that, through an understanding of normal bone healing events, we may be able to successfully inhibit resynostosis after surgical correction of craniosynostosis. The healing of bone fractures and surgically created defects has been rigorously studied. Several bone morphogenetic proteins (BMPs) have been extensively studied for their ability to induce bone formation, including BMP2, BMP4, and BMP7.(22,23) More importantly, BMPs have been shown to be expressed during normal bone healing, suggesting their involvement in the healing process.(24,25) Noggin has been identified as an extracellular antagonist to BMPs(26,27) and has been shown to be expressed along with BMP4 during normal bone healing.(28) Furthermore, Noggin has been shown to inhibit ectopic bone formation when delivered either systemically(29) or locally,(30) and has been used to inhibit membranous bone healing.(31) With regard to the cranial sutures, Noggin has been shown to inhibit normal suture fusion in the mouse, implicating a role for BMPs in initial suture fusion.(32) Because of the apparent role of BMP signaling in bone induction and in normal suture fusion, perturbation of BMP activity may lead to inhibition of bone healing. In an attempt to develop an adjunct to standard surgical techniques that improves postoperative outcomes, we hypothesized that local application of Noggin to the suturectomy site would inhibit resynostosis and improve craniofacial growth in a well-described rabbit model of human, nonsyndromic coronal suture synostosis.(7,20,33–38)

MATERIALS AND METHODS Rabbit model of craniosynostosis Previous studies have described the breeding demographics, pedigree analysis, and phenotypic variability of a colony of rabbits with coronal suture synostosis.(33,39) Briefly, the craniosynostosis that occurs in this colony varies from partial (delayed- or postgestational onset) to complete unilateral or bilateral coronal suture synostosis (early- or prenatal onset).(33,34,40) Early-onset synostosis in this model occurs around the 27th day of the 31-day rabbit gestation.(33,38) The primary synostotic event in these rabbits(34) leads to secondary deformations in the skull,(35) cranial base,(36) neurocapsule,(7) and brain.(38) Because of the similarity between the onset and progression of craniosynostosis in these rabbits compared with craniosynostosis that occurs in humans, this rabbit colony stands as the best animal model for human nonsyndromic craniosynostosis.

Sample Thirty-one New Zealand white rabbits with bilateral coronal suture synostosis (Fig. 1A) were obtained from the ongoing breeding colony of craniosynostotic rabbits housed at the University of Pittsburgh described above.(33,39,41) The rabbits were randomly assigned to three groups as follows: group 1, suturectomy with no treatment, which served as the surgical control group (n ⳱ 13); group 2, suturectomy with nonspecific BSA in a slow release collagen vehicle, which served as the protein control group (n ⳱ 8); group 3, suturectomy with Noggin protein in a slow release collagen vehicle, which served as the treatment group (n ⳱ 10). This protocol was approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.

Surgical technique After diagnosis of bilateral coronal suture synostosis, the synostosed coronal sutures were extirpated in 10-day-old rabbits using a strip suturectomy procedure described previously.(20,42) All rabbits were anesthetized with an intra-

1048 muscular injection (0.59 ml/kg rabbit body weight) of a solution containing 91% Ketaset (ketamine hydrochloride, 100 mg/ml) and 9% Rompun (xylazine, 20 mg/ml). The scalps were shaved, depilated, and prepared for surgery. The calvariae were exposed by a midline scalp incision, and the skin was reflected laterally to the supraorbital borders. Each synostosed coronal suture was identified, and a cutting burr was used to extirpate a 3-mm-wide strip of frontal and parietal bones, including the entire length and width of the coronal suture, in one piece (∼3 × 15-mm defects). Care was taken to preserve the meningeal (fibrous) layer of the dura and the regional vascularity. In rabbits in the suturectomy control group, only the suturectomy was performed; nothing was injected. The periosteal and skin incisions were closed with 4-0 Vicryl suture. In rabbits in the vehicle/protein control group, a 26-gauge needle was used to apply a dense (32.5 mg/ml) collagen vehicle (0.1 ml/suture; NeuColl, Campbell, CA, USA) that was mixed with BSA (Sigma) into the suturectomy site (10 ␮g BSA per defect; Figs. 1B and 1C). In the third group of defects, collagen mixed with Noggin protein (Sigma) was applied for a final concentration of 10 ␮g/defect. Next, a fine dental burr (0.5 mm) was used to make holes in the periosteum and bone for radiopaque amalgam markers. The holes were placed in quadrants, 2 mm anterior and posterior to the coronal, frontonasal, and lambdoidal sutures and 2 mm lateral to the sagittal and interfrontal sutures. These holes were packed with silver dental amalgam to serve as radiopaque markers for the radiographic analysis of postoperative craniofacial growth. All animals received postoperative intramuscular injections (2.5 mg/kg of rabbit body weight) of Baytril (Bayer, Shawnee Mission, KS, USA).

Data collection Somatic and skeletal growth: Longitudinal somatic (body weight), skeletal (third right metacarpal length from radiographs), and cephalometric growth data were recorded for all rabbits at 10, 25, 42, and 84 days of age. At 84 days of age, ∼90% of calvarial(2,18,33,43,44) and 90% of brain growth(38,45) are completed in rabbits. Lateral and dorsoventral radiographs of the head and front right paw were taken with the rabbits tranquilized with an intramuscular injection (10 mg/kg) of Ketaset (ketamine hydrochloride, 100 mg/ml). The heads were immobilized in a specially designed cephalostat, and a Phillips Oralix 70 dental X-ray unit was used at an exposure of 50 kV, 7 mA, a 0.17- to 0.50-s exposure time, and a tube-to-cassette distance held constant at 152 cm. Lateral and dorsoventral radiographs of the head were viewed on a light box, and the amalgam markers and landmarks under study were identified and traced. Craniofacial growth in a number of dimensions was assessed, including amalgam marker separation at the coronal suturectomy site, craniofacial length, and cranial vault shape. All measurements were taken blind as to rabbit group identity, and intraobserver, repeated measurement reliability was calculated (r ⳱ 0.901; p < 0.01) on a randomly drawn sample (10%) of rabbit cephalographs. Suturectomy site healing and intracranial volume: Serial CT scans of the rabbit heads were obtained at 10, 25, 42,

COOPER ET AL. and 84 days of age. A GE HiSpeed Advantage Scanner (DFOV ⳱ 24.0–18.0 cm; mA ⳱ 120–150; kV ⳱ 120) was used at a thickness of 1 mm to scan heads in the sagittal plane. The boundaries of the suturectomy sites were automatically traced, the healing defect site reconstructed in three dimensions, and the defect areas calculated. The endocortical boundaries of the cranial vault cavities were also traced both automatically and manually and reconstructed in three dimensions. Finally, the indirect intracranial volume (ICV) of each animal at each time-point was calculated. All calculations were performed with Allegro Software (ISG Technologies, Atlanta, GA, USA) on a Sun Workstation. All measurements were taken blind as to rabbit group identity, and intraobserver repeated measurement reliability was calculated (r ⳱ 0.936; p < 0.01) on a randomly drawn sample (20%) of the rabbit 3D-CT scans.

Statistical analysis Means and SD for suturectomy site areas, ICVs, body weights, third metacarpal lengths, and the craniofacial measurements were calculated and compared among groups using a 3 × 4 (group by age) two-way ANOVA with an unweighted means analysis. Significant intergroup differences at each age were assessed using the least significant differences multiple comparison test. All data were analyzed using SPSS 12.0 for windows (SPSS, Chicago, IL, USA). Differences were considered significant if p < 0.05.

Histological analysis At 84 days of age, all rabbits were killed by intravenous injection of pentobarbital (300 mg/kg rabbit body weight), and the defects were harvested for histological examination. The specimens were fixed, demineralized in 10% EDTA, and processed for paraffin sectioning. The specimens were sectioned in a sagittal plane in the middle of both the right and left coronal sutures at a thickness of 5 ␮m. Sections were stained with H&E for qualitative histological description.

RESULTS Mean body weight in all three groups changed in a linear fashion across age (Fig. 2A). Significant group (F ⳱ 5.83; p < 0.01) and age (F ⳱ 586.54; p < 0.001) main effects were noted. No significant group × age interaction effects (F ⳱ 1.20; not significant) were seen. Multiple comparison tests revealed that BSA-treated rabbits were significantly (p < 0.05) heavier than the other two groups at 42 days of age (Fig. 2A). Mean metacarpal length increased in a curvilinear fashion in all three groups (Fig. 2B). A significant age main effect (F ⳱ 161.42; p < 0.001) was noted. No significant group (F ⳱ 1.83; not significant) or group × age interaction effects (F ⳱ 0.61; not significant) were seen. Lateral cephalographs at 84 days of age showed still patent suturectomy sites, widened coronal suture amalgam markers, and slightly altered craniofacial skeletons in Noggin-treated rabbits compared with the two other groups (Fig. 3). Analysis of lateral cephalographs (Fig. 4A) showed that that Noggin-treated rabbits had greater marker separation distances at all postoperative intervals compared

NOGGIN INHIBITS RESYNOSTOSIS

FIG. 2. Somatic growth results. (A) Graph showing the changes in body weight over time (±SE). Note: there were no significant differences in body weight. (B) Graph showing third metacarpal length over time (±SE). No significant differences were found between groups.

with both control groups (Fig. 4B). Statistical analysis revealed significant group (F ⳱ 7.00; p < 0.001) and age (F ⳱ 23.38; p < 0.001) main effects. No significant group × age interaction effect was noted (F ⳱ 0.98; not significant). Multiple comparison tests revealed that coronal suture marker separation in Noggin-treated rabbits was significantly (p < 0.05) greater than suturectomy controls or BSAtreated control rabbits at all three postoperative ages (Fig. 4B). No significant difference in mean marker separation was noted between suturectomy controls or BSA-treated rabbits at any age. Mean craniofacial length in all three groups changed in a curvilinear fashion across age (Fig. 4C). Significant group (F ⳱ 2.39; p < 0.05) and age (F ⳱ 369.73; p < 0.001) main effects were noted. No significant group × age interaction effect (F ⳱ 0.81; not significant) was seen. Multiple comparison tests revealed that Noggin-treated

1049 rabbits had significantly (p < 0.05) greater craniofacial lengths than suturectomy controls or BSA-treated rabbits only at 84 days of age (Fig. 4C). Mean cranial vault shape indices decreased in a curvilinear fashion in all three groups over time (Fig. 4D). Noggin-treated rabbits showed greater indices than the other two groups at all three postoperative times. However, only a significant age main effect (F ⳱ 23.72; p < 0.001) was noted. No significant group (F ⳱ 0.59; not significant) or group by age interaction effects (F ⳱ 0.70; not significant) were seen (Fig. 4D). Suturectomy sites in both suturectomy control rabbits and rabbits treated with BSA showed rapid reossification through 84 days of age, as seen on the 3D-CT scan reconstructions (Fig. 5A). In contrast, suturectomy sites in rabbits treated with Noggin increased in size at 25 days of age and were more patent at 84 days of age (Fig. 5A). Healing in all groups was most evident along the lateral margins of the defect, with any remaining defect being evident along the midline (Fig. 5A). Mean suturectomy site area in both suturectomy control rabbits and rabbits treated with BSA showed similar reossification rates that decreased to ∼30% of the original defect area by 84 days of age (Fig. 5B). In contrast, mean suturectomy site areas in rabbits treated with Noggin increased to ∼120% of original defect size at 25 days of age and had ∼60% of the original defect area remaining at 84 days of age (Fig. 5B). It is interesting to note that suturectomy site areas in Noggin-treated rabbits paralleled the two other control groups from 25 to 84 days of age (Fig. 5B). Statistical analysis revealed significant group (F ⳱ 23.29; p < 0.001) and age (F ⳱ 31.75; p < 0.001) main effects. No significant group × age interaction effect was noted (F ⳱ 0.19; not significant), showing that defect area was changing in a parallel fashion among groups over time. Multiple comparison tests revealed that Noggin-treated defects were significantly (p < 0.05) larger than suturectomy controls or BSA-treated control defects at all time-points after surgery (Fig. 5B). No significant differences in mean defect area were noted between suturectomy controls or BSA-treated rabbits at any age. ICV in all three groups showed similar curvilinear changes from 10 to 84 days of age (Fig. 5C). Noggin-treated rabbits had greater ICVs from 25 to 84 days of age (Fig. 5C). Statistical analysis revealed significant group (F ⳱ 5.15; p < 0.01) and age (F ⳱ 180.74; p < 0.001) main effects. No significant group × age interaction effect was noted (F ⳱ 1.30; not significant), showing that ICV was changing in a parallel fashion among groups over time. Multiple comparison tests revealed that ICV in Noggintreated rabbits was significantly (p < 0.05) greater than suturectomy controls or BSA-treated control rabbits only at 84 days of age (Fig. 5C). No significant differences in mean ICV were noted between suturectomy controls or BSAtreated rabbits at any age. Gross microscopic analysis revealed that, at 84 days of age, the suturectomy control group had complete wound healing, with extensive reossification and resynostosis of the suturectomy sites (Fig. 6, suturectomy control). Most of the original suturectomy sites were completely obliterated (Fig. 6, suturectomy control). The BSA-treated group had incomplete wound healing with some bony bridges. Most

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FIG. 3. Lateral cephalographs of select rabbits. Lateral cephalographs were used to measure the separation of radioopaque markers placed in the calvaria at the time of suturectomy (10 days of age). Note that the Noggin-treated (NOG) rabbits showed large areas of radiolucent defects even at 84 days of age (arrows) compared with suturectomy control (SC) and BSA-treated (BSA) animals.

FIG. 4. Cephalometric data. (A) Diagram depicting the radiographic landmarks used to collect cephalometric data: marker separation ⳱ CS, craniofacial length ⳱ MOPPR, cranial vault shape index ⳱ (ALS-SOS)/ (OP-FE). (B) Coronal suture marker separation (±SE) shows that Noggin-treated defects allowed for significantly increased marker separation compared with suturectomy and BSA-treated defects (p < 0.05). (C) Mean craniofacial length (±SE) was increased in Noggin-treated animals at 84 days of age compared with suturectomy and BSAtreated controls (p < 0.05). (D) Cranial vault shape indices (±SE) were found to be greater in Noggin-treated animals. Analysis revealed that the differences were not significant.

sections through these defects showed some fibrous tissue in the suturectomy site (Fig. 6, BSA). The Noggin-treated group had incomplete wound healing, with immature bone formation (Fig. 6, Noggin).

DISCUSSION Craniosynostosis can result from several identified genetic mutations, including FGFR1, FGFR2, MSX, and

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FIG. 5. CT reconstructions and defect healing data. (A) 3D reconstructions of serial CT scans performed on suturectomy control, BSA-treated, and Noggin-treated rabbits at 10, 25, 42, and 84 days of age. Noggin seemed to inhibit healing of suturectomy sites. (B) Analysis of defect area (±SE) showed that Noggin-treated defects were significantly larger at every time-point after surgery (25, 42, and 84 days of age; p < 0.05). (C) 3D analysis of intracranial volume (±SE) showed that Noggin treatment increased intracranial volume significantly at 84 days of age (p < 0.05).

TWIST. For the nonsyndromic craniosynostoses, the genetic and/or epigenetic causes remain largely unknown. Much of the recent research in craniosynostosis has centered on the elucidation of the molecular mechanisms that lead to either normal(46–48) or premature(49–52) suture fusion. This line of research has led to a better understanding of the complexity of the molecular interactions that regulate bone formation and suture development and maintenance, although it has not, as yet, been used to develop biologically based therapies to reduce the occurrence of postoperative resynostosis or to improve surgical outcomes. One correlate that is common to all types of craniosynostoses, regardless of genetic or environmental cause, is an overgrowth of bone. Normal sutures remain patent, or devoid of bony bridging, to allow for the growth of the developing brain. The fusion of sutures occurs by the ossification of the normally patent suture through the process of osteogenic differentiation. We believe that the regulation of bone overgrowth should be the target of any therapy developed to improve treatment of craniosynostosis if a single therapy is to be useful to all patients presenting with craniosynostosis. Because BMP2, -4, and -7 have been found to be potent bone-inducing proteins that are expressed during normal bone healing,(25) we hypothesized that BMP function may be instrumental in the occurrence of resynostosis

in a rabbit model of human nonsyndromic craniosynostosis. Noggin was chosen to test this hypothesis because of its ability to inhibit the activity of BMP2, -4, and -7.(27) It was observed that Noggin treatment was effective in inhibiting bone healing and delaying suturectomy site resynostosis in the rabbit model, evidenced by the long-term persistence of defects in the calvaria up to 84 days after treatment. It must be noted that Noggin treatment did not maintain a defect that was the same size or shape of the original suturectomy. In fact, the healing that occurred at the lateral margins of Noggin-treated defects was reossified and resynostosed. These data suggest that, although Noggin was able to inhibit bone healing in this model, the delivery technique must be improved to inhibit bone formation in a more controlled manner. The data also suggest that Noggin, delivered once, within a slowly resorbing collagen gel, can have long-term effects on craniofacial growth in this model. There was an initial increase in the size of the suturectomy site in the Noggintreated group. This increase is caused by the rapid expansion of the skull in the anteroposterior direction after surgical release of the fused suture that was the cause for the initial anteroposterior shortening of the skull. Although this skull expansion may also be occurring in the BSA and untreated control groups (evidenced by the similar ICV

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FIG. 6. Histology at 84 days of age. Histological analysis revealed that sections from suturectomy control and BSA-treated defects showed almost complete defect healing at 84 days of age, whereas sections from Noggin-treated defects showed large areas of fibrous tissue within the defect at 84 days of age. These qualitative data support the radiographic data showing larger, unhealed defects in the Noggin-treated group compared with the other groups.

measurements at 25 and 42 days of age; Fig. 5C), the amount of bone healing obscures this fact. It is important to note here that the increase in defect size did not seem to occur by destruction or resorption of bone, evidenced by the normal morphology in the bone and soft tissues surrounding the suturectomy site (Fig. 6). The inhibition of bone healing by Noggin therapy occurred primarily in the first 15 days after surgery (up to 25 days of age), followed by the resumption of bone healing at rates similar to control defects. Notice that the graph of reossification (Fig. 5B) shows that lines connecting 25, 42, and 84 days in the Noggin-treated group closely parallel the lines in the two control groups. The only difference is that the Noggin-treated group’s defects increased in size between 10 and 25 days of age, starting their normal healing from a different defect size than the two control groups. The resumption of normal healing after 25 days of age in the Noggin-treated group may reflect the loss of Noggin function caused by its release from the collagen gel over time. The collagen vehicle used here was a highly purified bovine type I collagen that was chosen because of its bio-

COOPER ET AL. compatibility, its low antigenicity (from the removal of the antigenic N- and C-terminal domains), its superior handling properties (semiviscous [injectable] at room temperature and gel at 37°C), and its slow degradation rate (∼6 m to 1 yr in vivo).(53) Although the protein was added to this slowly resorbing collagen gel, it is likely that most of the protein released from the gel over the first 2 wk in vivo. In fact, recent studies using this dense collagen gel as a vehicle for growth factor delivery has shown that growth factors remained active up to 15 days in culture with cells.(54) This type of release would account for the normal healing that is noticed after 25 days of age (15 days after surgery). The hypothesis regarding the 15-day duration of Noggin activity is also supported by the marker separation data. In this study, it was observed that the markers in the Noggintreated group were significantly farther apart at 25 days of age compared with the control groups and not significantly different at the later time-points. The marker separation data reflect the occurrence of resynostosis (healing) along the lateral aspects of the defects, causing a reduction in the rate of marker separation. The data presented here show that a single dose of Noggin can have lasting effects on bone formation and improve ICV and craniofacial growth in synostotic rabbits almost to levels similar to wildtype rabbits, despite the finding that the therapy had a limited temporal activity. In rabbits, 90% of the total brain growth is completed by 84 days of age,(38,45) whereas, in humans, it takes nearly 6 yr to complete 90% of brain growth.(55) Therefore, therapies developed for use in humans must have a much longer period of effect than those studied in our rabbit model of simple, nonsyndromic craniosynostosis. For this reason, our group is working on testing several delivery mechanisms for protein-based and gene-based therapies. Although the genetic mutation responsible for craniosynostosis in this rabbit model has not been identified yet, data obtained from this model suggest that postoperative resynostosis probably uses one of a number of highly conserved signaling pathways. This should make the design and development of other biologically based therapies much easier, especially if the goal is to interrupt downstream signaling and reduce osteogenesis at the surgical site and not treat the primary genetic etiology of craniosynostosis. Noggin-based therapy may represent a particularly useful means for the inhibition of postoperative resynostosis for two reasons. First, Noggin therapy, similar to any biologically based adjunct to surgery, may improve surgical outcomes while at the same time allow for minimal surgical intervention. Small craniectomies, placed where sutures normally exist, may be able to remain patent through the inhibition of bone formation in that region using Nogginbased therapies, thus allowing for more normal brain and craniofacial growth postoperatively. Second, by targeting BMP signaling, it may be possible to sidestep the direct molecular cause of the original synostosis. BMPs are potent inducers of bone formation. Interruption of BMP signaling should inhibit the osteogenic differentiation of cells regardless of their genetic background. Although much work needs to be done with respect to understanding the role of BMPs in craniosynostosis, their role in bone healing is well

NOGGIN INHIBITS RESYNOSTOSIS understood. It is likely that the inhibition of BMP signaling may be the upstream event that can nullify many of the downstream events that are associated with the known mutations that lead to craniosynostosis. Most importantly, this study showed that Noggin treatment may have some effect on the abnormal healing patterns that are exhibited by individuals (both rabbit and human) with craniosynostosis.

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ACKNOWLEDGMENTS

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This study was supported by the Jorge Posada Foundation (BJC and MPM) and NIH/NIDCR 2 R01 DE 01342006 (JH).

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Address reprint requests to: Gregory M Cooper, PhD Division of Pediatric Plastic Surgery Children’s Hospital of Pittsburgh 3705 Fifth Avenue Pittsburgh, PA 15213, USA E-mail: [email protected] Received in original form January 8, 2007; revised form March 30, 2007; accepted April 6, 2007.