Interleukin-10 Does Not Augment Osseous Regeneration in the Scarred Calvarial Defect Achieved with Low-Dose Biopatterned BMP2.

BACKGROUND: Large calvarial defects represent a major reconstructive challenge, as they do not heal spontaneously. Infection causes inflammation and scarring, further reducing the healing capacity of the calvaria. Bone morphogenetic protein-2 (BMP2) has been shown to stimulate osteogenesis but has significant side effects in high doses. BMP2 has not been tested in combination with antiinflammatory cytokines such as interleukin-10. METHODS: Sixteen New Zealand White rabbits underwent 15 × 15-mm flap calvarectomies. The flap was incubated in Staphylococcus aureus and replaced, and infection and scarring were allowed to develop. The flap was subsequently removed and the wound débrided. A 15 × 15-mm square of acellular dermal matrix biopatterned with low-dose BMP2, interleukin-10, or a combination was implanted. Computed tomographic scans were taken over 42 days. Rabbits were then killed and histology was performed. RESULTS: Defects treated with BMP2 showed significantly (p < 0.05) greater osseous regeneration than untreated controls. Interleukin-10 did not significantly augment the healing achieved with BMP2, and interleukin-10 alone did not significantly increase healing compared with controls. Histology showed evidence of bone formation in defects treated with BMP2. Untreated controls and defects treated with interleukin-10 alone showed only fibrous tissue in the defect site. CONCLUSIONS: Low-dose BMP2 delivered directly to the scarred calvarial defect augments bony healing. Interleukin-10 at the dose applied did not significantly augment healing alone or in combination with BMP2. Healing had not finished at 42 days and analysis at later time points or the use of higher doses of BMP2 may yield greater healing.

Molecular Analyses in a Rabbit Model of Craniosynostosis: Likely Exclusion of Known Candidate Genes as the Loci of Origin.

OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. We work with a naturally occurring rabbit model of CS with an undefined etiology. Known causes of coronal CS were evaluated to identify potential associations with CS in the rabbit. DESIGN: Candidate genes were sequenced in control New Zealand White (NZW) rabbits (n = 4) and synostotic NZW rabbits (n = 4). Variants were identified by alignment using Clustal Omega. OUTCOME MEASURES: Single nucleotide variants (SNVs) were classified according to phenotypic associations and predicted impact on protein structure. Human correlates were identified in the database of single nucleotide polymorphisms (dbSNP). RESULTS: A total of 21 SNVs were identified in the 10 genes examined. Variant classification and inheritance patterns are inconsistent with causality. CONCLUSIONS: The genetic basis for disease in the CS rabbit likely involves novel loci and is not associated with known causes of coronal synostosis.

Reconstruction of a Calvarial Wound Complicated by Infection: Comparing the Effects of Biopatterned Bone Morphogenetic Protein 2 and Vascular Endothelial Growth Factor.

Bone morphogenetic protein 2 (BMP2) bioprinted on biological matrix induces osseous regeneration in large calvarial defects in rabbits, both uncomplicated and scarred. Healing in unfavorable defects scarred from previous infection is decreased due in part to the lack of vascularity. This impedes the access of mesenchymal stem cells, key to osseous regeneration and the efficacy of BMP2, to the wound bed. The authors hypothesized that bioprinted vascular endothelial growth factor (VEGF) would augment the osseous regeneration achieved with low dose biopatterned BMP2 alone. Thirteen New Zealand white rabbits underwent subtotal calvariectomy using a dental cutting burr. Care was taken to preserve the underlying dura. A 15 mm × 15 mm flap of bone was cut away and incubated in a 1 × 108 cfu/mL planktonic solution of S aureus before reimplantation. After 2 weeks of subsequent infection the flap was removed and the surgical wound debrided followed by 10 days of antibiotic treatment. On postoperative day 42 the calvarial defects were treated with acellular dermal matrix bioprinted with nothing (control), VEGF, BMP2, BMP2/VEGF combined. Bone growth was analyzed with serial CT and postmortem histology. Defects treated with BMP2 (BMP2 alone and BMP2/VEGF combination) showed significantly greater healing than control and VEGF treated defect (P < 0.5). Vascular endothelial growth factor treated defect demonstrated less healing than control and VEGF/BMP2 combination treatments achieved less healing than BMP2 alone though these differences were nonsignificant. Low dose BMP2-patterned acellular dermal matrix improves healing of scarred calvarial defects. Vascular endothelial growth factor at the doses applied in this study failed to increase healing.

Genetic associations and phenotypic heterogeneity in the craniosynostotic rabbit.

Craniosynostosis (CS) is a disorder that involves the premature ossification of one or more cranial sutures. Our research team has described a naturally occurring rabbit model of CS with a variable phenotype and unknown etiology. Restriction-site associated DNA (RAD) sequencing is a genomic sampling method for identifying genetic variants in species with little or no existing sequence data. RAD sequencing data was analyzed using a mixed linear model to identify single nucleotide polymorphisms (SNPs) associated with disease occurrence and onset in the rabbit model of CS. SNPs achieving a genome-wide significance of p ≤ 5 x 10-8 were identified on chromosome 2 in association with disease occurrence and on chromosomes 14 and 19 in association with disease onset. Genotyping identified a coding variant in fibroblast growth factor binding protein 1 (FGFBP-1) on chromosome 2 and a non-coding variant upstream of integrin alpha 3 (ITGA3) on chromosome 19 that associated with disease occurrence and onset, respectively. Retrospective analysis of patient data revealed a significant inverse correlation between FGFBP-1 and ITGA3 transcript levels in patients with coronal CS. FGFBP-1 and ITGA3 are genes with roles in early development that warrant functional study to further understand suture biology.

The influence of suturectomy on age-related changes in cerebral blood flow in rabbits with familial bicoronal suture craniosynostosis: A quantitative analysis.

BACKGROUND: Coronal suture synostosis is a condition which can have deleterious physical and cognitive sequelae in humans if not corrected. A well-established animal model has previously demonstrated disruptions in intracranial pressure and developmental abnormalities in rabbits with congenital craniosynostosis compared to wild type rabbits. OBJECTIVE: The current study aimed to measure the cerebral blood flow (CBF) in developing rabbits with craniosynostosis who underwent suturectomy compared to those with no intervention and compared to wild type rabbits. METHODS: Rabbits with early onset coronal suture synostosis were assigned to have suturectomy at 10 days of age (EOCS-SU, n = 15) or no intervention (EOCS, n = 18). A subset of each group was randomly selected for measurement at 10 days of age, 25 days of age, and 42 days of age. Wild type rabbits (WT, n = 18) were also randomly assigned to measurement at each time point as controls. Cerebral blood flow at the bilateral hemispheres, cortices, thalami, and superficial cortices was measured in each group using arterial spin-labeling MRI. RESULTS: At 25 days of age, CBF at the superficial cortex was significantly higher in EOCS rabbits (192.6 ± 10.1 mL/100 mg/min on the left and 195 ± 9.5 mL/100 mg/min on the right) compared to WT rabbits (99.2 ± 29.1 mL/100 mg/min on the left and 96.2 ± 21.4 mL/100 mg/min on the right), but there was no significant difference in CBF between EOCS-SU (97.6 ± 11.3 mL/100 mg/min on the left and 99 ± 7.4 mL/100 mg/min on the right) and WT rabbits. By 42 days of age the CBF in EOCS rabbits was not significantly different than that of WT rabbits. CONCLUSION: Suturectomy eliminated the abnormally increased CBF at the superficial cortex seen in EOCS rabbits at 25 days of age. This finding contributes to the evidence that suturectomy limits abnormalities of ICP and CBF associated with craniosynostosis.

Maternal environment and craniofacial growth: geometric morphometric analysis of mandibular shape changes with in utero thyroxine overexposure in mice.

An estimated 3% of US pregnancies are affected by maternal thyroid dysfunction, with between one and three of every 1000 pregnancies being complicated by overactive maternal thyroid levels. Excess thyroid hormones are linked to neurological impairment and excessive craniofacial variation, affecting both endochondral and intramembranous bone. Using a geometric morphometric approach, this study evaluates the role of in utero thyroxine overexposure on the growth of offspring mandibles in a sample of 241 mice. Canonical variate analysis utilized 16 unilateral mandibular landmarks obtained from 3D micro-computed tomography to assess shape changes between unexposed controls (n = 63) and exposed mice (n = 178). By evaluating shape changes in the mandible among three age groups (15, 20 and 25 days postnatal) and different dosage levels (low, medium and high), this study found that excess maternal thyroxine alters offspring mandibular shape in both age- and dosage-dependent manners. Group differences in overall shape were significant (P < 0.001), and showed major changes in regions of the mandible associated with muscle attachment (coronoid process, gonial angle) and regions of growth largely governed by articulation with the cranial base (condyle) and occlusion (alveolus). These results compliment recent studies demonstrating that maternal thyroxine levels can alter the cranial base and cranial vault of offspring, contributing to a better understanding of both normal and abnormal mandibular development, as well as the medical implications of craniofacial growth and development.

Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model. DESIGN: Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits. RESULTS: Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1. CONCLUSIONS: These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.

Rescue of Premature Coronal Suture Fusion with TGF-ß2 Neutralizing Antibody in Rabbits with Delayed-Onset Synostosis.

OBJECTIVES: An overexpression of Tgf-ß2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-ß2 may help rescue fusing sutures and restore normal growth. The present study was designed to test this hypothesis. DESIGN: Twenty-eight New Zealand White rabbits with delayed-onset coronal synostosis had radiopaque markers placed on either side of the coronal sutures at 10 days of age. The rabbits were randomly assigned to: (1) sham control rabbits (n = 10), (2) rabbits with control IgG (100 µg/suture) delivered in a collagen vehicle (n = 9), and (3) rabbits with Tgf-ß2 neutralizing antibody (100 µg/suture) delivered in a collagen vehicle (n = 9). Longitudinal growth data were collected at 10, 25, 42, and 84 days of age. Sutures were harvested at 84 days of age for histomorphometry. RESULTS: Radiographic analysis showed significantly greater ( P < .05) coronal suture marker separation, craniofacial length, cranial vault length, height, shape indices, cranial base length, and more lordotic cranial base angles in rabbits treated with anti-Tgf-ß2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-ß2 antibody at 84 days of age had patent and significantly ( P < .05) wider coronal sutures and greater sutural area compared to controls. CONCLUSIONS: These data support our hypothesis that antagonism of Tgf-ß2 may rescue fusing coronal sutures and facilitate craniofacial growth in this rabbit model. These findings also suggest that cytokine therapy may have clinical significance in infants with progressive postgestational craniosynostosis.

Molecular Analysis of Ephrin A4 and Ephrin B1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis (CS) has a prevalence of approximately 1 in every 2000 live births and is characterized by the premature fusion of one or more cranial sutures. Failure to maintain the cell lineage boundary at the coronal suture is thought to be involved in the pathology of some forms of CS. The Ephrin family of receptor tyrosine kinases consists of membrane-bound receptors and ligands that control cell patterning and the formation of developmental boundaries. Mutations in the ephrin A4 (EFNA4) and ephrin B1 (EFNB1) ligands have been linked to nonsyndromic CS and craniofrontonasal syndrome, respectively, in patient samples. We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if EFNA4 or EFNB1 could be the loci of the causal mutation in this unique animal model. Sequencing of EFNA4 and EFNB1 was performed using templates obtained from wild-type (n = 4) and craniosynostotic (n = 4) rabbits. No structural coding errors were identified in either gene. A single-nucleotide transversion was identified in one wild-type rabbit within the third intron of EFNA4. These data indicate that the causal locus for heritable CS in this rabbit model is not located within the structural coding regions of either EFNA4 or EFNB1.

Genetic Homozygosity and Phenotypic Variability in Craniosynostotic Rabbits.

BACKGROUND: Craniosynostosis ranges in severity from single suture involvement with prenatal onset to multiple suture involvement with postnatal onset. The present study was designed to test the hypothesis that increasing homozygosity may be responsible for more severe phenotypic expression by examining the relationship between inbreeding and phenotypic expression in synostotic rabbits. METHODS: Data were obtained from 173 litters and 209 rabbits with familial craniosynostosis. Five distinct phenotypes were identified (normal n = 62; unicoronal delayed onset synostosis (DOS) n = 47; bicoronal DOS n = 21; unicoronal early onset synostosis (EOS) n = 26, and bicoronal EOS n= 53). Wright's coefficients of inbreeding (CI) were calculated using CompuPed software. Radiographs were taken at 10, 25, 42, 84, and 126 days of age to assess coronal suture, craniofacial, and skeletal growth. The relationship between CI and growth data was assessed using correlation coefficients. RESULTS: Mean CIs ranged from 15.68 (±2.22) in normal rabbits to 25.89 (±5.03) in bicoronal DOS, to 36.29 (±2.10) in unicoronal EOS to 42.85 (±2.10) in bicoronal EOS rabbits. Significant differences were noted among groups (F = 11.48; P < .001). Significant negative correlations were noted between CI and sutural and craniofacial growth at 25 (r = -.45, P < .001; and r = -.66, P < .001) through 126 (r = -.40, P < .001 and r = -.46, P < .001) days of age. CONCLUSIONS: While the synostotic phenotype is inherited in an autosomal dominant fashion in these rabbits, increasing homozygosity is associated with more severely affected phenotypes. These findings suggest that an accumulation of additional, modifier genes may determine the severity of the synostotic phenotype in rabbits.

Application of Laser Capture Microdissection to Craniofacial Biology: Characterization of Anatomically Relevant Gene Expression in Normal and Craniosynostotic Rabbit Sutures.

OBJECTIVE: Fusion of the cranial sutures is thought to depend on signaling among perisutural tissues. Mapping regional variations in gene expression would improve current models of craniosynostosis. Laser capture microdissection (LCM) isolates discrete cell populations for gene expression analysis. LCM has rarely been used in the study of mineralized tissue. This study sought to evaluate the potential use of LCM for mapping of regional gene expression within the cranial suture. DESIGN: Coronal sutures were isolated from 10-day-old wild-type and craniosynostotic (CS) New Zealand White rabbits, and LCM was used to isolate RNA from the sutural ligament (SL), osteogenic fronts (OF), dura mater, and periosteum. Relative expression levels for Fibroblast Growth Factor 2 (FGF2), Fibroblast Growth Factor Receptor 2 (FGFR2), Transforming Growth Factor Beta 2 (TGFß-2), Transforming Growth Factor Beta 3 (TGFß-3), Bone Morphogenetic Protein 2 (BMP-2), Bone Morphogenetic Protein 4 (BMP-4), and Noggin were determined using quantitative real-time PCR. RESULTS: A fivefold increase in TGFß2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFß-3 was detected within the OF of CS animals. Noggin expression was increased by 10-fold within the CS SL, but reduced by 13-fold within the CS dura. Reduced expression of FGF2 was observed within the CS SL and dura, whereas increased expression of FGFR2 was observed within the CS SL. Reduced expression of BMP-2 was observed in the CS periosteum, and elevated expression of BMP-4 was observed in the CS SL and dura. CONCLUSIONS: LCM provides an effective tool for measuring regional variations in cranial suture gene expression. More precise measurements of regional gene expression with LCM may facilitate efforts to correlate gene expression with suture morphogenesis and pathophysiology.

Simonart's Band: Its Effect on Cleft Classification and Recommendations for Standardized Nomenclature.

OBJECTIVE: Accurate classification of cleft lip plays an important role in communication, treatment planning, and comparison of outcomes across centers. Although there is reasonable consensus in defining cleft types, the presence of Simonart's band can make classification challenging. Our objective was to survey cleft care providers to determine what all consider to be Simonart's band, how its presence effects cleft lip classification, and to provide recommendations for standardized nomenclature. DESIGN: A multiple-choice survey was e-mailed to 1815 members of the American Cleft Palate-Craniofacial Association, assessing each respondent's definition of Simonart's band and its effect on cleft classification. Cleft classification was drawn from the ICD system diagnosis billing codes. Descriptive analysis was performed. RESULTS: Three hundred seventy-three providers completed the survey (20.5% response), the majority of whom were surgeons (61.5%); 87.1% agreed with the definition that a Simonart's band is "any soft tissue bridge located at the base of the nostril or more internally, between the segmented ridges." However, only 41.8% felt that the presence of a Simonart's band rendered a cleft lip incomplete; 54.4% felt that an alveolar cleft was the defining difference between a complete and an incomplete cleft lip. When asked to define the child with a cleft involving the upper lip that extends into the naris but interrupted by a soft tissue bridge located only at the base of the nostril or more internally, without a cleft of the alveolar ridge and palate, 61.4% classified this as an incomplete cleft lip, 32.7% as a complete cleft lip, and 5.9% as an unspecified cleft lip. CONCLUSIONS: Responses revealed wide discrepancy in the classification of cleft phenotypes and in the interpretation of the significance of anatomical components in the classification of a cleft lip. We discuss the difficulty in aligning classification based on unclear definition of terms and variable anatomic parameters. We highlight this issue in the face of a need for comparability in clinical evidence-based practices. To ensure precision and uniformity in cleft classification, we recommend that use of the term "Simonart's band" be abandoned while incorporating a notation of the integrity of the nasal sill into the LAHSHAL system. We propose a uniform definition of incomplete versus complete cleft lip, wherein a cleft lip will be classified as complete in the presence or absence of narrow bands of tissue present at the base of the nasal sill or more internally.

Repair of a Complicated Calvarial Defect: Reconstruction of an Infected Wound With rhBMP-2.

BACKGROUND: Management of the previously infected craniofacial defect remains a significant clinical challenge, posing obstacles such as wound healing complications, lack of donor site availability, and predisposition to failure of the repair. Optimal therapy would reconstruct like with like, without donor site morbidity. The purpose of this study was to compare the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2)-mediated bone regeneration with the current standard of autologous bone graft for repair of previously infected calvarial defects. METHODS: Nineteen adult New Zealand white rabbits underwent subtotal calvariectomy. Bone flaps were inoculated with Staphylococcus aureus and replanted. After 1 week of infection, bone flaps were removed, and wounds were debrided, followed by 10 days of antibiotic treatment. After 6 weeks, animals underwent scar debridement followed by definitive reconstruction in 1 of 4 groups: empty control (n = 3), vehicle control (buffer solution on absorbable collagen sponge [ACS], n = 3), autologous bone graft (n = 3), or rhBMP-2 repair (rhBMP-2/ACS, n = 10). Animals underwent computed tomography imaging at 0, 2, 4, and 6 weeks postoperatively, followed by euthanization and histological analysis. Percent healing was determined by 3-dimensional analysis. A (time × group) 2-way analysis of variance was performed on healing versus treatment group and postoperative time. RESULTS: At 6 weeks postoperatively, rhBMP-2/ACS and autologous bone graft resulted in 93% and 68% healing, respectively, whereas the empty and vehicle control treatment resulted in 27% and 26% healing (P < 0.001). Histologically, compared to autologous bone graft, bone in the rhBMP-2/ACS group was more cellular and more consistently continuous with wound margins. CONCLUSIONS: The rhBMP-2 therapy is effective in achieving radiographic coverage of previously infected calvarial defects.

The influence of surgical correction on white matter microstructural integrity in rabbits with familial coronal suture craniosynostosis.

OBJECT Craniosynostosis is a condition in which one or more of the calvarial sutures fuses prematurely. In addition to the cosmetic ramifications attributable to premature suture fusion, aberrations in neurophysiological parameters are seen, which may result in more significant damage. This work examines the microstructural integrity of white matter, using diffusion tensor imaging (DTI) in a homogeneous strain of rabbits with simple, familial coronal suture synostosis before and after surgical correction. METHODS After diagnosis, rabbits were assigned to different groups: wild-type (WT), rabbits with early-onset complete fusion of the coronal suture (BC), and rabbits that had undergone surgical correction with suturectomy (BC-SU) at 10 days of age. Fixed rabbit heads were imaged at 12, 25, or 42 days of life using a 4.7-T, 40-cm bore Avance scanner with a 7.2-cm radiofrequency coil. For DTI, a 3D spin echo sequence was used with a diffusion gradient (b = 2000 sec/mm(2)) applied in 6 directions. RESULTS As age increased from 12 to 42 days, the DTI differences between WT and BC groups became more pronounced (p < 0.05, 1-way ANOVA), especially in the corpus callosum, cingulum, and fimbriae. Suturectomy resulted in rabbits with no significant differences compared with WT animals, as assessed by DTI of white matter tracts. Also, it was possible to predict to which group an animal belonged (WT, BC, and BC-SU) with high accuracy based on imaging data alone using a linear support vector machine classifier. The ability to predict to which group the animal belonged improved as the age of the animal increased (71% accurate at 12 days and 100% accurate at 42 days). CONCLUSIONS Craniosynostosis results in characteristic changes of major white matter tracts, with differences becoming more apparent as the age of the rabbits increases. Early suturectomy (at 10 days of life) appears to mitigate these differences.

Transforming growth factor beta 1 augments calvarial defect healing and promotes suture regeneration.

BACKGROUND: Repair of complex cranial defects is hindered by a paucity of appropriate donor tissue. Bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 1 (TGFß1) have been shown separately to induce bone formation through physiologically distinct mechanisms and potentially improve surgical outcome for cranial defect repair by obviating the need for donor tissue. We hypothesize that a combination of BMP2 and TGFß1 would improve calvarial defect healing by augmenting physiologic osteogenic mechanisms. METHODS/RESULTS: Coronal suturectomies (3×15 mm) were performed in 10-day-old New Zealand White rabbits. DermaMatrix™ (3×15mm) patterned with four treatments (vehicle, 350 ng BMP2, 200 ng TGFß1, or 350 ng BMP2+200 ng TGFß1) was placed in suturectomy sites and rabbits were euthanized at 6 weeks of age. Two-dimensional (2D) defect healing, bone volume, and bone density were quantified by computed tomography. Regenerated bone was qualitatively assessed histologically. One-way analysis of variance revealed significant group main effects for all bone quantity measures. Analysis revealed significant differences in 2D defect healing, bone volume, and bone density between the control group and all treatment groups, but no significant differences were detected among the three growth factor treatment groups. Qualitatively, TGFß1 treatment produced bone with morphology most similar to native bone. TGFß1-regenerated bone contained a suture-like tissue, growing from the lateral edge of the defect margin toward the midline. Unique to the BMP2 treatment group, regenerated bone contained lacunae with chondrocytes, demonstrating the presence of endochondral ossification. CONCLUSIONS/SIGNIFICANCE: Total healing in BMP2 and TGFß1 treatment groups is not significantly different. The combination of BMP2+TGFß1 did not significantly increase bone healing compared with treatment with BMP2 or TGFß1 alone postoperatively at 4 weeks. We highlight the potential use of TGFß1 to regenerate calvarial bone and cranial sutures. TGFß1 therapy significantly augmented bony defect healing at an earlier time point when compared with control, regenerated bone along the native intramembranous ossification pathway, and (unlike BMP2 alone or in combination with TGFß1) permitted normal suture reformation. We propose a novel method of craniofacial bone regeneration using low-dose, spatially controlled growth factor therapies to minimize potentially harmful effects while maximizing local bioavailability and regenerating native tissues.

Bone morphogenetic protein 2­mediated mandible reconstruction successfully heals bony defects but inhibits concurrent inferior alveolar nerve grafting: a rabbit experimental model.

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) has been used to reconstruct mandibular defects. An elegant addition to this reconstruction method would be incorporation of a nerve graft wrapped in a BMP-2 carrier to reconstitute the inferior alveolar nerve (IAN) and restore sensation to the lower face. We developed a rabbit model to determine the effect BMP-2 has on nerve regeneration following neurorrhaphy. METHODS: An inferior border mandibulectomy was created in 16 adult New Zealand white rabbits. The IAN was protected, divided, and repaired with either primary neurorrhaphy or reverse autografts. Bone defects were treated with no treatment controls (n = 2), absorbable collagen sponge (ACS) (vehicle controls) (n = 7), and ACS soaked in BMP-2 (treatment group) (n = 7). Animals underwent computed tomography (CT) 2 days and 6 weeks postoperatively. The percent bone defect healing was calculated using Amira 3D imaging software. At 6 weeks, IANs were harvested mesial to the reconstruction and were evaluated with toluidine blue histology to identify myelinated axons. Reconstructed mandible segments were evaluated with micro-CT and hematoxylin-eosin histology. RESULTS: Bone morphogenetic protein 2-treated animals demonstrated significantly more bone healing than did the ACS and empty defect groups (82%, 38%, 44%, respectively; P < 0.01). One hundred percent of ACS-treated nerves (n = 4) demonstrated axon regrowth, whereas only 25% of BMP-2-treated nerves (n = 4) did. Micro-CT and histology showed BMP-2 caused bone growth around the IAN, but regenerated bone infiltrated the repair site and created a physical barrier to axon growth. CONCLUSIONS: Bone morphogenetic protein 2 can successfully heal bone defects in the rabbit mandible, but ectopic bone growth can inhibit IAN recovery after repair. Level of Evidence: Not gradable.

Cleft Palate-Craniofacial Journal 50th anniversary editorial board commentary: anatomy, basic sciences, and genetics--then and now.

To celebrate the 50th year of the Cleft Palate-Craniofacial Journal we look back to where we started in 1964 and where we are now, and we speculate about directions for the future in a "Then and Now" editorial series. This editorial examines changing trends and perspectives in anatomical, basic science, and genetic studies published in this 50-year interval. In volume 1 there were 45 total papers, seven (16%) of which were peer-reviewed basic science and genetic articles published: four in anatomy, three in craniofacial biology, and none in genetics. In contrast, in volume 50, of 113 articles there were 47 (42%) peer-reviewed basic science and genetic articles published: 30 in anatomy, five in craniofacial biology, and 12 in genetics. Topical analysis of published manuscripts then and now reveal that similar topics in anatomy and craniofacial biology are still being researched today (e.g., phenotypic variability, optimal timing of surgery, presurgical orthopedics, bone grafting); whereas, most of the more recent papers use advanced technology to address old questions. In contrast, genetic publications have clearly increased in frequency during the last 50 years, which parallels advances in the field during this time. However, all of us have noticed that the more "cutting-edge" papers in these areas are not being submitted for publication to the journal, but instead to discipline-specific journals. Concerted efforts are therefore indicated to attract and publish these cutting-edge papers in order to keep the Cleft Palate-Craniofacial Journal in the forefront of orofacial cleft and craniofacial anomaly research and to provide a valuable service to American Cleft Palate-Craniofacial Association members.

Cloning of TgfßR1 and TgfßR2 and Likely Exclusion as Loci of Origin in a Rabbit Craniosynostotic Model.

OBJECTIVE: To determine whether TgfßR1 or TgfßR2 cause the craniosynostotic phenotype in a rabbit model of nonsyndromic craniosynostosis. DESIGN: Full-length TgfßR1 and TgfßR2 cDNAs were sequenced and real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to measure TgfßR1 and TgfßR2 transcripts in sutural tissue from wild type (WT) and craniosynostotic (CS) rabbits. Single nucleotide polymorphisms (SNP) were identified within TgfßR1 and TgfßR2 and were assayed for segregation with disease phenotype in 22 craniosynostotic animals. RESULTS: No structural mutations in TgfßR1 and TgfßR2 were identified in the craniosynostotic rabbits. Real-time RT-PCR quantification of TgfßR1 and TgfßR2 mRNA showed no significant difference in TgfßR1 expression between CS and WT animals, while TgfßR2 showed 50% elevation in the CS animals compared to WT (P < .05). SNP analysis within the TgfßR1 and TgfßR2 genes suggested that neither locus is linked to the craniosynostotic phenotype because no allelic combination showed any specific correlation with disease phenotype for either TgfßR1 or TgfßR2. CONCLUSIONS: Our data indicate that the craniosynostotic phenotype in this rabbit model does not arise from any structural mutation in TgfßR1 or TgfßR2, and SNP analysis also likely excludes these genes more broadly as the site of causative mutation.

Ectocranial suture fusion in primates: pattern and phylogeny.

Patterns of ectocranial suture fusion among Primates are subject to species-specific variation. In this study, we used Guttman Scaling to compare modal progression of ectocranial suture fusion among Hominidae (Homo, Pan, Gorilla, and Pongo), Hylobates, and Cercopithecidae (Macaca and Papio) groups. Our hypothesis is that suture fusion patterns should reflect their evolutionary relationship. For the lateral-anterior suture sites there appear to be three major patterns of fusion, one shared by Homo-Pan-Gorilla, anterior to posterior; one shared by Pongo and Hylobates, superior to inferior; and one shared by Cercopithecidae, posterior to anterior. For the vault suture pattern, the Hominidae groups reflect the known phylogeny. The data for Hylobates and Cercopithecidae groups is less clear. The vault suture site termination pattern of Papio is similar to that reported for Gorilla and Pongo. Thus, it may be that some suture sites are under larger genetic influence for patterns of fusion, while others are influenced by environmental/biomechanic influences.

Molecular Analysis of Twist1 and FGF Receptors in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

Craniosynostosis is the premature fusion of the cranial vault sutures. We have previously described a colony of rabbits with a heritable pattern of nonsyndromic, coronal suture synostosis; however, the underlying genetic defect remains unknown. We now report a molecular analysis to determine if four genes implicated in human craniosynostosis, TWIST1 and fibroblast growth factor receptors 1-3 (FGFR1-3), could be the loci of the causative mutation in this unique rabbit model. Single nucleotide polymorphisms (SNPs) were identified within the Twist1, FGFR1, and FGFR2 genes, and the allelic patterns of these silent mutations were examined in 22 craniosynostotic rabbits. SNP analysis of the Twist1, FGFR1, and FGFR2 genes indicated that none were the locus of origin of the craniosynostotic phenotype. In addition, no structural mutations were identified by direct sequence analysis of Twist1 and FGFR3 cDNAs. These data indicate that the causative locus for heritable craniosynostosis in this rabbit model is not within the Twist1, FGFR1, and FGFR2 genes. Although a locus in intronic or flanking sequences of FGFR3 remains possible, no direct structural mutation was identified for FGFR3.