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.

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.

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 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.

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.

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.

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.

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.

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: as related to cranial volume and dental eruption.

BACKGROUND: Timing of calvarial suture fusion is important in primate ontogeny. Ages at death are difficult to assess especially for museum collections. METHODS: 1550 skulls of Hominoid, Hylobatidae, Macaca and Papio were observed for fusion. Calvarial expansion (early) and dental eruption (late) were utilized as indicators of ontogeny. Homogeneity of slopes and ANOVA were used to determine differences in timing of fusion. RESULTS: For calvarial growth the great apes all showed small levels of calvarial suture remodeling prior to full calvarial expansion. For dental eruption, Homo and Macaca share a common pattern of fusion in late adulthood. The other species show early remodeling. Papio was observed to have distinct patterns for suture fusion progression. CONCLUSIONS: Thus, suture fusion progression although influenced by evolutionary changes in the robusticity of the craniofacial skeleton can be modeled by the phylogeny among this group. Overall, Homo appears to have a distinct pattern of delayed suture fusion progression.

Novel model of calvarial defect in an infected unfavorable wound: reconstruction with rhBMP-2. Part II.

BACKGROUND: Animal models of bone reconstruction have shown recombinant human bone morphogenetic protein 2 (rhBMP-2) to be an effective therapy in the acute calvarial defect wound. The purpose of this study was to compare the effectiveness of rhBMP-2 in a rabbit model of an unfavorable scarred calvarial wound with the criterion standard of autograft. METHODS: Nineteen adult New Zealand white rabbits underwent subtotal calvariectomy. After 6 weeks of healing and normal scar formation, these animals underwent reoperation for scar debridement and assignment to 1 of 4 therapeutic groups. Animals were assigned to an empty control group (no treatment, n = 3), vehicle control group (neutral buffered solution on an absorbable collagen sponge [ACS], n = 3), surgical control group (cryopreserved autograft, n = 3), or an experimental treatment group (rhBMP-2 on an ACS, n = 10). All animals underwent computed tomography imaging at 0, 2, 4, and 6 weeks after secondary reconstructive surgery. At 6 weeks, all animals were killed, and the defects were examined histologically. Percentage of healing of each defect was determined, and a 4 × 3 mixed-model analysis of variance was performed on healing as a function of time and therapy. RESULTS: Based on measures of defect radiopacity, the treatment group (rhBMP-2/ACS) and surgical control group (autograft) were statistically equivalent with 98% and 83% healing, respectively, at 6 weeks. The empty control and vehicle control groups were inferior to the treatment group (rhBMP-2/ACS) and surgical control (autograft) groups at each timepoint (P < 0.05). Histologically, bone in the surgical control (autograft) group was less trabecular and less cellular than the bone formed in the experimental treatment group (rhBMP-2/ACS). CONCLUSIONS: Compared with historical controls, rhBMP-2 therapy was as effective in reconstructing calvarial defects in the unfavorable scarred wound as in the acute favorable calvarial wound. When compared with cryopreserved autograft, rhBMP-2-regenerated bone showed equal defect coverage and similar bone thickness with varying bony architecture.

Tissue interactions between craniosynostotic dura mater and bone.

BACKGROUND: Cells within the dura mater have been implicated in the determination of suture patency and fusion. Craniosynostosis (CS), the premature fusion of 1 or more of the cranial sutures, could result from abnormal control over the differentiation of osteoprogenitor cells from the dura mater. This study tested whether dura mater cells derived from rabbits with congenital CS were different from cells derived from normal rabbits and investigated the effects that CS dura mater had on osteogenic differentiation in vitro and in vivo. METHODS: Cells were derived from the dura mater from wild-type rabbits (WT; n = 23) or CS rabbits (n = 16). Cells were stimulated with bone morphogenetic protein 4, and alkaline phosphatase (ALP) expression and cell proliferation were assessed. Dura mater-derived cells were also cocultured with primary rabbit bone-derived cells, and ALP was assessed. Finally, interactions between the dura mater and overlying tissues were manipulated in vivo. RESULTS: Craniosynostotic dura mater-derived cells proliferated faster than did WT cells but were not more ALP positive. Coculture experiments showed that CS dura mater cells induced increased ALP activity in CS bone-derived cells, but not in WT bone-derived cells. In vivo experiments showed that a physical barrier successfully inhibited dura mater-derived osteogenesis. CONCLUSIONS: Coculture of CS bone- and CS dura mater-derived cells evoked an abnormal phenotype in vitro. Covering the CS dura mater led to decreased bone formation in vivo. Further investigations will focus on the signaling molecules involved in the communication between these 2 CS tissue types in vitro and in vivo.

Relaxin does not rescue coronal suture fusion in craniosynostotic rabbits.

OBJECTIVES: Craniosynostosis affects 1 in 2000 to 3000 live births and may result in craniofacial and neural growth disturbances. Histological data have shown that thick collagenous bundles are present in the sutural ligament, which may tether the osteogenic fronts, resulting in premature fusion. The hormone relaxin has been shown to disrupt collagen fiber organization, possibly preventing craniosynostosis by relaxing the sutural ligament and allowing osteogenic fronts to separate normally and stay patent. This study tested this hypothesis with a rabbit model of delayed-onset coronal suture synostosis. METHODS: A total of 18 New Zealand White rabbits with craniosynostosis were randomly assigned to one of three groups: sham control, protein control (BSA), relaxin treatment. After initial diagnosis, sham surgery, BSA, or relaxin was delivered to the fusing coronal suture in a slow-release (56-day) collagen vehicle. Longitudinal radiographs and body weights were collected at 10, 25, 42, and 84 days of age, and sutures were harvested for histology. RESULTS: Relaxin-treated animals had more disorganized intrasuture content than control groups. These specimens also appeared to have relatively wider sutures ectocranially. There were no significant differences in relaxin-treated animals for all craniofacial growth measures, or suture separation compared with controls. CONCLUSIONS: These data do not support our initial hypothesis that the use of relaxin may rescue sutures destined to undergo premature suture fusion. These findings suggest that collagen fiber arrangement may not be important for suture fusion. This protein therapy would not be clinically useful for craniosynostosis.

Timing of ectocranial suture activity in Gorilla gorilla as related to cranial volume and dental eruption.

Research has shown that Pan and Homo have similar ectocranial suture synostosis patterns and a similar suture ontogeny (relative timing of suture fusion during the species ontogeny). This ontogeny includes patency during and after neurocranial expansion with a delayed bony response associated with adaptation to biomechanical forces generated by mastication. Here we investigate these relationships for Gorilla by examining the association among ectocranial suture morphology, cranial volume (as a proxy for neurocranial expansion) and dental development (as a proxy for the length of time that it has been masticating hard foods and exerting such strains on the cranial vault) in a large sample of Gorilla gorilla skulls. Two-hundred and fifty-five Gorilla gorilla skulls were examined for ectocranial suture closure status, cranial volume and dental eruption. Regression models were calculated for cranial volumes by suture activity, and Kendall's tau (a non-parametric measure of association) was calculated for dental eruption status by suture activity. Results suggest that, as reported for Pan and Homo, neurocranial expansion precedes suture synostosis activity. Here, Gorilla was shown to have a strong relationship between dental development and suture activity (synostosis). These data are suggestive of suture fusion extending further into ontogeny than brain expansion, similar to Homo and Pan. This finding allows for the possibility that masticatory forces influence ectocranial suture morphology.

Comparative microcomputed tomography and histological study of maxillary pneumatization in four species of new world monkeys: the perinatal period.

In anthropoid primates, it has been hypothesized that the magnitude of maxillary sinus growth is influenced by adjacent dental and soft tissue matrices. Relatively, little comparative evidence exists for the perinatal period when secondary pneumatization is at its earliest stages in some primates. Here, dental and midfacial variables were studied in a perinatal sample of four anthropoid primates, including three callitrichines (Leontopithecus, Saguinus, and Callithrix) and Saimiri boliviensis. In the latter species, the maxillary recess (the ontogenetic precursor to a "true" maxillary sinus) does not undergo secondary pneumatization. Using histological methods and micro-computed tomography, midfacial and dental dimensions and radiographic hydroxyapatite density of tooth cusps were measured. The distribution of osteoclasts and osteoblasts was also documented. Kruskal-Wallis's one-way analysis of variance tests indicates significant (P < 0.05) differences among groups for dental and midfacial measurements. In particular, the posterior maxillary dentition is relatively larger and more mineralized in Saimiri compared to the callitrichines. At posterior dental levels, Saimiri has the lowest palatonasal index [interdental (palatal) width/width of the nasal cavity] and highest bizygomatic-interorbital index. Distribution of osteoclasts indicates that the inferomedial surfaces of the orbits are resorptive in perinatal Saimiri, whereas, in all callitrichines, these surfaces are depository. Taken together, these findings suggest that pneumatization in Saimiri is suppressed by an inward growth trajectory of the orbits, relatively large posterior dentition, and a correspondingly compressed nasal region.