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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.

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.

Objectives: An overexpression of Tgf-β2 leads to calvarial hyperostosis and suture fusion in individuals with craniosynostosis. Inhibition of Tgf-β2 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-β2 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-β2 antibody than in controls at 42 and 84 days of age. Histologically, rabbits treated with anti-Tgf-β2 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-β2 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.

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.

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.

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 (TGFβ-2), Transforming Growth Factor Beta 3 (TGFβ-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 TGFβ2 expression was detected in the CS SL relative to wild type, whereas 152-fold less TGFβ-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.

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.

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.

Objective: To determine whether TgfβR1 or TgfβR2 cause the craniosynostotic phenotype in a rabbit model of nonsyndromic craniosynostosis., Design: Full-length TgfβR1 and TgfβR2 cDNAs were sequenced and real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to measure TgfβR1 and TgfβR2 transcripts in sutural tissue from wild type (WT) and craniosynostotic (CS) rabbits. Single nucleotide polymorphisms (SNP) were identified within TgfβR1 and TgfβR2 and were assayed for segregation with disease phenotype in 22 craniosynostotic animals., Results: No structural mutations in TgfβR1 and TgfβR2 were identified in the craniosynostotic rabbits. Real-time RT-PCR quantification of TgfβR1 and TgfβR2 mRNA showed no significant difference in TgfβR1 expression between CS and WT animals, while TgfβR2 showed 50% elevation in the CS animals compared to WT (P < .05). SNP analysis within the TgfβR1 and TgfβR2 genes suggested that neither locus is linked to the craniosynostotic phenotype because no allelic combination showed any specific correlation with disease phenotype for either TgfβR1 or TgfβR2., Conclusions: Our data indicate that the craniosynostotic phenotype in this rabbit model does not arise from any structural mutation in TgfβR1 or TgfβR2, and SNP analysis also likely excludes these genes more broadly as the site of causative mutation.

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.

Objective: Overexpression of transforming growth factor-beta 2 has been associated with craniosynostosis and resynostosis following surgery. We examined the effects of localized transforming growth factor-beta 2 inhibition on craniofacial phenotype in rabbits with craniosynostosis., Design: Twenty-five New Zealand white rabbits with bilateral coronal craniosynostosis were divided into three treatment groups: (1) suturectomy control (n=8); (2) suturectomy with nonspecific, control immunoglobulin G antibody (n=6); and (3) suturectomy with anti-transforming growth factor-beta 2 antibody (n=11). At 10 days of age, a coronal suturectomy was performed on all rabbits. The sites in groups 2 and 3 were immediately filled with a slow-resorbing collagen gel mixed with either immunoglobulin G or anti-transforming growth factor-beta 2 antibody. Computed tomography scans of each rabbit were acquired at ages 10, 25, and 84 days. Craniofacial landmarks were collected from three-dimensional computed tomography reconstructions, and growth and form were compared among the three groups., Results: Rabbits treated with anti-transforming growth factor-beta 2 antibody differed in form at 84 days of age compared with suturectomy control rabbits, specifically in the snout and posterior neurocranium. Growth in some areas of the skull was greater in rabbits from the anti-transforming growth factor-beta 2 group than in suturectomy control rabbits, but not significantly greater than in IgG control rabbits., Conclusions: We find support for the hypothesis that transforming growth factor-beta 2 inhibition alters adult form, but these changes do not appear to be localized to the suturectomy region. Slight differences in form and growth between the two control groups suggest that the presence of the collagen vehicle itself may affect skull growth.

Objective: To determine if there is an anatomic basis for subepithelial orbicularis oris muscle defects by directly comparing ultrasonographic images of the upper lip with corresponding histological sections obtained from cadavers., Methods: Ultrasound was performed on the upper lips of previously frozen, unpreserved cadaver heads (n = 32), followed by dissection and sectioning of the upper lips. The ultrasound sequences were scored by a panel of raters, classifying the orbicularis oris muscle as negative, positive, or unknown for the presence of an orbicularis oris discontinuity (subepithelial defect). Based on ultrasound, six lip specimens were chosen for histological sectioning, were stained with hematoxylin-eosin and Gomori trichrome stain, and were evaluated with light microscopy at low (8x) and intermediate (100x) magnification., Results: One cadaver was scored positive for an orbicularis oris muscle discontinuity based on ultrasound; whereas, the remaining cadavers were scored negative. Of the cadavers with negative ultrasound scores, two were noted to have orbicularis oris muscles with "irregular" features (e.g., excessive localized thinning or asymmetry). From histology, the area of discontinuity as visualized on the positively scored ultrasound was characterized by both disorganized orbicularis oris muscle fibers and excess connective tissue within the muscle belly. In contrast, the localized thinning observed on some of the negatively scored ultrasounds was not confirmed by histology., Conclusions: Abnormalities of the orbicularis oris muscle visualized by ultrasound have an anatomic basis as revealed through histology. Ultrasound is a useful tool for noninvasively identifying discontinuities of the orbicularis oris muscle.

Objective: Various causal mechanisms of familial nonsyndromic craniosynostosis have been presented. One hypothesis suggests that overproduction of bone at the suture is the primary origin of craniosynostosis, which affects brain and cranial growth secondarily through altered intracranial pressure (Primary Suture Fusion Model). Other hypotheses suggest that decreased cranial base growth or abnormal brain growth are the primary cause of craniosynostosis (Cranial Base, Brain Parenchyma Models, respectively). This study was designed to investigate which model best describes neurocranial changes associated with craniosynostosis in a rabbit model through multivariate path analysis., Design: Serial magnetic resonance imaging scans and intracranial pressure measurements were obtained at 10, 25, and 42 days of age from 18 rabbits: six controls, six with delayed-onset synostosis, and six with early-onset synostosis. Five variables were collected from each rabbit: calvarial thickness at the affected suture, cranial base length, brain volume, cerebrospinal fluid volume, and intracranial pressure. This data set was used to test causal pathway relationships generated by the proposed models. Goodness of fit was measured by experimental group for each model., Results: Primary Suture Fusion Model best explained the variables in both delayed-onset and early-onset synostotic rabbits (Goodness of fit = 93%, 97%, respectively). Cranial Base Model (Goodness of fit = 94%) best explained the data in control rabbits., Conclusion: Results suggest that the primary site of craniosynostosis in craniosynostotic rabbits is most likely the synostosed suture. Other cranial vault anomalies are most likely secondary compensatory changes. Results of the present study may provide insight regarding the causal pathway of craniosynostosis.

The Pittsburgh Oral-Facial Cleft study was begun in 1993 with the primary goal of identifying genes involved in nonsyndromic orofacial clefts in a variety of populations worldwide. Based on the results from a number of pilot studies and preliminary genetic analyses, a new research focus was added to the Pittsburgh Oral-Facial Cleft study in 1999: to elucidate the role that associated phenotypic features play in the familial transmission patterns of orofacial clefts in order to expand the definition of the nonsyndromic cleft phenotype. The purpose of this paper is to provide a comprehensive review of phenotypic features associated with nonsyndromic orofacial clefts. These features include fluctuating and directional asymmetry, non-right-handedness, dermatoglyphic patterns, craniofacial morphology, orbicularis oris muscle defects, dental anomalies, structural brain and vertebral anomalies, minor physical anomalies, and velopharyngeal incompetence.

Objective: To describe the expression patterns of the various transforming growth factor-beta (Tgf-beta) isoforms, known to be involved in suture development, in the perisutural tissues of rabbits with naturally occurring craniosynostosis and relate such differential expression to the pathogenesis of premature suture fusion., Method: Twenty-one coronal sutures were harvested from six wild-type control New Zealand White rabbits and five rabbits with familial coronal suture synostosis at 25 days of age for histomorphometric and immunohistochemical analyses. Tgf-beta isoform immunoreactivity was assessed using indirect immunoperoxidase procedures with specific antibodies., Results: Synostosed sutures had significantly (p <.01) greater bone area and relatively more osteoblasts and osteocytes in the osteogenic fronts, compared with wild-type sutures. Tgf-beta isoform immunoreactivity showed differential staining patterns between wild-type and synostosed perisutural tissues. In wild-type sutures, Tgf-beta1 and Tgf-beta3 immunoreactivity was significantly (p <.001) greater than Tgf-beta2 staining in all perisutural tissues. In synostosed sutures, the opposite pattern was observed, with Tgf-beta2 immunoreactivity significantly (p <.001) greater than Tgf-beta1 and Tgf-beta3 in the osteogenic fronts, dura mater, and periosteum., Conclusions: Findings from this study suggest that an overexpression of Tgf-beta2, either in isolation or in association with an underexpression of Tgf-beta1 and Tgf-beta3, may be related to premature suture fusion (craniosynostosis) in this pathological rabbit model. These abnormal expression patterns may be involved in premature suture fusion either through increased cell proliferation, decreased apoptosis of the osteoblasts or both at the osteogenic fronts.

Objective: Clinical studies have shown both abnormal and normal mental and psychomotor development in patients with craniosynostosis. However, a number of confounding variables make study comparisons difficult. For these reasons, the present study describes early neuromotor development in an homogeneous rabbit model of craniosynostosis., Design: Fifty-three newborn New Zealand white rabbit kits were used: 13 were wild-type, normal control rabbits; 23 had delayed-onset coronal suture synostosis (onset is approximately 57 to 74 days post conception); and 17 had early-onset coronal suture synostosis (onset is approximately 21 to 25 days post conception). All rabbits were observed individually and blindly in an open field, daily for 2 minutes, from birth through the first 14 days of life. The first day of emergence of 10 different mature behaviors and developmental events (in developmental order of appearance: falling, righting, cliff avoidance, first sign of fur, body elevation, head elevation, circling, dragging, eye opening, and hopping) was recorded for each kit. Daily activity levels (grid crossing), and body weights were also recorded., Results: Significant group (p <.05) differences were observed in 9 of 11 measures. Both synostosed groups had significantly (p <.05) accelerated onset of behavior in 8 of 9 measures, compared with wild-type controls. The early-onset synostosis group had significantly (p <.05) accelerated onset in five of eight measures, compared with wild-type controls, and three of eight measures, compared with the delayed-onset synostosis group., Conclusions: Synostotic rabbits showed precocious neuromotor development possibly through frontal lobe constrictions and altered brain activity from increased intracranial pressure, although primary genetic effects cannot be ruled out.

Objective: To determine whether antibody perturbation of Tgf-beta, delivered in a collagen gel, could inhibit cranial suture fusion., Design: Attachment and proliferation of osteoblasts cultured on a collagen gel with or without anti-Tgf-beta2 antibody were determined by AlamarBlue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, collagen gel with and without anti-Tgf-beta2 antibody was injected subperiosteally over the posterior frontal suture of postnatal day 15 rat calvariae. A quantitative analysis of suture fusion was used to measure suture bridging in histological serial sections at various time points., Results: Attachment and proliferation for cells cultured on collagen gel with anti-Tgf-beta2 antibody were similar to collagen gel controls. Although proliferation was lower than on tissue culture plastic, cells treated with anti-Tgf-beta2 antibody maintained an osteoblastic morphology. After 7, 10, and 15 days in organ culture, anti-Tgf-beta2 antibody treatment caused a reduction in the percent bridging of posterior frontal sutures, compared with controls. Sutures exposed to anti-Tgf-beta2 antibody and fibroblast growth factor-2 concurrently did not show an inhibition of bony bridging., Conclusions: These results support previous reports suggesting a role for Tgf-beta2 in cranial suture fusion. In cell culture the collagen gel, both with and without anti-Tgf-beta2 antibody, promoted similar osteoblast attachment, proliferation, and osteoblastic morphology. In organ culture anti-Tgf-beta2 antibody was delivered in a bioactive state via a collagen gel to inhibit cranial suture fusion. Also, the results suggest that the inductive effect of fibroblast growth factor-2 is not dependent on Tgf-beta2 activity. Together, these results provide further support for the role of Tgf-beta2 in cranial suture fusion.

Objective: Craniosynostosis has been associated with fibroblast growth factors (FGFs) and their receptors. The purpose of this study was to quantitatively determine the effect of FGF2 on rat calvarial osteoblasts and a rat cranial suture formation model., Design: Fetal rat calvarial osteoblasts were cultured with and without FGF2. Cell attachment and proliferation was determined by alamar Blue dye assay and cell morphology by toluidine-blue staining. In rat calvarial organ culture, postnatal day 15 rat calvariae with dura mater were placed in serum-free media with and without FGF2. A unique quantitative analysis of suture fusion was developed by obtaining measurements of suture bridging in histological serial sections at progressive stages of fusion., Results: Attachment for cells treated with FGF2 was similar to control. In contrast, proliferation was higher for cells treated with FGF2 while maintaining an osteoblastic morphology. After 5 days in organ culture, FGF2-treated posterior frontal sutures showed a dramatic increase in fusion, compared with untreated controls. This increased fusion was maintained throughout days 7 and 10 in culture. Also, fusion was enhanced on the dural side of the suture, as is normally observed in vivo, and the normal tissue architecture was maintained., Conclusions: These results indicate that FGF2 can promote rat osteoblast attachment and normal cell morphology as well as induce cell proliferation. In calvarial organ culture, FGF2 treatment produced an enhanced suture fusion. These results provide further support for a critical role for FGF2 in cranial suture development. These studies also present a new quantitative approach to evaluating the effect of suture-perturbing growth factors on cranial suture fusion.

Objective: To retrospectively study prenatal ultrasound images of patients with craniosynostosis to determine the extent to which prenatal diagnosis is possible., Method: Prenatal ultrasound images of 19 patients with postnatally diagnosed metopic or coronal suture craniosynostosis were retrospectively reviewed. The 26 ultrasound examinations obtained were compared with normal images and tables of gestation., Results: It was not possible to diagnose craniosynostosis in the first trimester. In the second trimester, Kleeblattschädel was diagnosed at 20.5 weeks. A multilobular shape to the skull and diastasis of the frontotemporal suture was identified. In a second child with Kleeblattschädel, the cephalic index was above normal 86.4 (normal range 70 to 86), and the head circumference to abdominal circumference was increased. In the third trimester, the head shape deformation was more obvious. Brachycephaly diagnosis was made in the second trimester. In Crouzon syndrome the hypertelorism was identified at 19.9 weeks. Plagiocephaly was diagnosed at 21.4 weeks. In trigonocephaly the reduced cephalic index was noted at 18.8 weeks. In the third trimester, the deformity was diagnosed in all cases., Conclusion: No diagnosis of craniosynostosis was made in the first trimester. In the second trimester, it was possible to diagnose Kleeblattschädel, trigonocephaly, brachycephaly (bilateral coronal suture craniosynostosis), and plagiocephaly (unilateral coronal suture craniosynostosis) in nine of the examinations. In the third trimester and at term, it was possible to diagnose previously listed conditions from six of the seven examinations obtained. Kleeblattschädel was suspected during original examination. A total of 15 examinations of 26 were correctly diagnosed during this investigation.