Relationships between growth at the sutures of the rabbit calvarium

The present study was designed to elucidate the relationships between growth increments at the cranial vault sutures in rabbits. Thirteen male New Zealand white rabbits were followed regularly from age 31 to 142 days using a roentgen stereophotogrammetric system. Spherical tantalum markers were implanted into the nasal, frontal, and parietal bones, and implant stability was checked at each stereo examination. Problems with instability were encountered only in the nasal bones. Registered growth rates conformed to our previous investigations. High correlations were observed between the following areas; the coronal suture to the frontonasal suture, the first principal component of the neurocranial suture group to the frontonasal suture, and the principal component of the craniofacial suture group to the coronal suture. Remaining relationships demonstrated dispersion to various extents. The findings indicate that there seems to exist a basic mutual dependence between neural and facial skeletal growth, as well as complex covariations between the various sutures of the rabbit calvarium.     

The correlation between craniofacial and long bone growth: an experimental investigation in normal rabbits

The present study was undertaken to elucidate the relationships between craniofacial and long-bone growth. Nine male New Zealand white rabbits received spherical tantalum bone markers in the tibial epiphyses and in the nasal, frontal, and parietal bones. The animals were followed from 30 to 143 days of age. Growth changes were calculated with a roentgen stereometric system, and the results statistically evaluated. Except for the final interval when all variables varied at random, high correlations between tibial and frontonasal or coronal sutural growth were demonstrated; and the respective linear regression lines were homogeneously assembled. The relationship between the tibia and the sagittal suture displayed great variations between individual animals as well as between the suture's parts, although growth at the interfrontal suture was clearly correlated to tibial growth upon exclusion of the time factor. The first principal component of the three neurocranial sutures was calculated and seemed accurately correlated to long-bone growth. The present study concluded that growth at the frontonasal and coronal sutures normally seems to parallel general somatic development, while growth at the sagittal suture appears individually displaced in time. Nevertheless, when the principal component of the combination of the coronal suture and the neurocranial section of the sagittal suture was computed, this was highly correlated to body growth.     

Craniofacial suture stenosis: morphologic effects

Craniofacial anomalies, such as Apert's and Crouzon's syndromes, are presumed to be related to premature growth arrest of cranial base growth sites. However, premature growth arrest at cranial vault sutures in animals appears to play a causative role in the development of cranial deformities characteristic of single-suture, or simple, craniosynostosis in humans. To study the possible causative role of cranial vault and other (interface) suture stenoses on the development of craniofacial deformity, a vault suture and an interface suture between the cranial vault and facial skeleton were simultaneously immobilized. Thirty-one New Zealand White rabbits at 9 days of age underwent implantation of dental amalgam growth markers adjacent to cranial vault and facial sutures. In the experimental group (n = 15), methylcyanoacrylate adhesive was applied over the coronal (vault) and frontonasal (interface suture between vault and facial skeleton) sutures to immobilize them. The remaining 16 animals served as sham-treated controls. All animals underwent serial radiographic cephalometry to document growth effects in the cranial vault, cranial base, and facial skeleton. Application of adhesive resulted in statistically significant (p less than 0.05) reduction in growth at the coronal and frontonasal sutures. This was accompanied by an overall significant reduction in neurocranial vault length during the first 30 days of development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skull base expansion: craniofacial effects

In order to determine what effect the anterior cranial base has on the developing craniofacial skeleton, mechanical expansion of the growth of one segment of the anterior cranial base was performed. New Zealand white rabbits were used for this study. A sham-treated group (n = 16) underwent implantation of dental amalgam markers to either side of the frontonasal, coronal, and lambdoid sutures at 9 days of age to serve as markers of vault growth. The experimental group (n = 7) underwent the same marker placement at 9 days of age, but, in addition, at 30 days of age these animals underwent placement of a mechanical spring, unilaterally, at the frontosphenoid suture. A second control group (n = 8) underwent the same exposure of the frontosphenoid suture, but the spring was laid only on the surface of the bone. All animals were followed by radiographic cephalometry at 9, 30, 60, and 90 days of age. The experimental group demonstrated statistically significant expansion of the cranial base and ipsilateral coronal suture. The midface skeletal dimensions were unchanged by spring distraction of the cranial base. These findings indicate that cranial base sutural growth can be manipulated mechanically and that growth changes can be attained secondarily in the cranial vault skeleton.     

Growth restriction of cranial sutures in the fetal lamb causes deformational changes, not craniosynostosis

Newborns with in utero cranial vault molding can present with severe forms of plagiocephaly. Intrauterine constraint has been proposed as one cause for craniosynostosis. The purpose of this experiment was to investigate whether rigid plate fixation across a fetal cranial suture, representing a severe form of growth restriction in utero, would lead to cranial suture fusion in a fetal lamb model. Six fetal lambs at 85 to 95 days gestation (term = 145 days) underwent laparotomy, hysterotomy, fetal coronal scalp incision, and miniplate screw fixation across the right coronal suture in utero. Two unoperated twins and four unoperated age-matched lambs were used as controls (n = 12). Animals were killed at both 4 and 8 weeks postoperatively. Fetal head analysis consisted of gross examination, photography, basilar and lateral radiographs, and three-dimensional computed tomographic scans. Cranial suture analysis consisted of imaging by computed tomographic scan (axial and sagittal cuts) and histology of experimentally plated coronal sutures, contralateral nonplated coronal sutures and twin control coronal sutures. Gross examination, radiographs, and three-dimensional computed tomographic analysis of heads with cranial suture plating showed ipsilateral forehead flattening, contralateral forehead bossing, superiorly displaced ipsilateral orbital rim, anterolateral projection of ipsilateral malar eminence, and anterior position of the ipsilateral ear point compared with the contralateral side of the same animal and normal controls. There was no change in nasal root, chin point, or predentition occlusal plane. Although analysis of the plated coronal sutures by computed tomographic scans showed diminished width or even stenosis, the histology revealed narrowed but patent experimental coronal sutures at 4 and 8 weeks. Contralateral, nonplated coronal sutures were not only patent, but widened compared with normal control sutures. This finding may have represented compensatory changes in the contralateral coronal suture caused by growth restriction at the plated suture. These data demonstrate that intrauterine growth restriction across a cranial suture caused by compression plate fixation resulted in deformational skull changes, not craniosynostosis. In addition, these data strongly support a role for in utero positional molding secondary to growth restriction in the maternal pelvis as a cause for nonsynostotic plagiocephaly seen in newborns.     

Nasal capsular cartilage is required for rat transpalatal suture morphogenesis

In the cranial vault, suture morphogenesis occurs when the growing cranial bones approximate and overlap or abut one another. Patency of developing sutures is regulated by the underlying dura mater. Once cranial sutures form, bone growth proceeds from the sutures in response to growth signals from the rapidly expanding neurocranium. Facial sutures do not develop in contact with the dura mater. It was therefore hypothesized that facial suture morphogenesis and bone growth from facial sutures are regulated by tissues with an equivalent role to the dura mater. The present study was designed to test this hypothesis by characterizing the morphology and growth factor expression in developing transpalatal (TP) sutures and their surrounding tissues, and then assessing the role of the overlying nasal capsular (NC) cartilages in maintaining suture patency. TP sutures develop as overlapping sutures, similar to cranial coronal sutures, and expression of Tgf-betas in TP sutures was similar to their distribution in cranial coronal sutures. To establish whether NC cartilages play a role in regulating TP suture morphogenesis, fetal rat TP sutures were cultured with associated attached NC cartilages or with NC cartilages removed. Sutures cultured for upward of 5 days with intact NC cartilages remained patent and maintained their cellular and fibrous components. However, in the absence of NC cartilages, the cellular nature of the sutures was not maintained and they became progressively acellular, with bony bridging across the suture. This finding is similar to that for cranial vault sutures cultured in the absence of dura mater, indicating that NC cartilages play an equivalent role to dura mater in maintaining the patency of developing sutures. These studies indicate that tissue interactions likely regulate morphogenesis of all cranial and facial sutures.     

Kinematics of cranial vault growth in rabbits

To characterize mathematically the spatial rearrangement of cranial vault bones of the rabbit during growth, a longitudinal study was undertaken from age 4-20 weeks. Initially, at least three nonlinear tantalum bone markers were implanted in the parietal, frontal, and the combined nasal bones. Thereafter, the animals were followed regularly with roentgen stereophotogrammetrical analysis. The parietal bones were found to rotate laterally upward (3 degrees), while the frontal bones rotated downward (2 degrees) relative to their contralaterals. The frontal bones rotated rostrally upward (12 degrees) and outward (3 degrees) as well as laterally downward (5 degrees) in relation to the parietal bones. Due to the morphology of the rabbit head, the examination positioning used in this study, and the direction of the growth process, growth at the coronal suture correlated fairly well with longitudinal axis translations; but the growth at the frontonasal suture relative to the frontal bones was directed about 45 degrees downward. This points to the importance of the bone-marker positioning, so that their connecting line is directed along the axis of growth. Also, this approach makes it possible to obtain new information on the development and treatment of craniofacial aberrations.     

Maxillary volume growth in craniosynostosis

Craniosynostosis, and in particular, craniofacial dysostosis, exhibits abnormalities of the nasomaxillary complex in form, position, and development. The aim of this study was to quantitatively assess the volumetric maxillary abnormality in patients at the time of initial diagnosis of craniosynostosis and to make comparisons with a "normal" reference range for maxillary volumes throughout childhood. The technique of segmentation was applied to preoperative computed tomographic head scans obtained in 31 children (14 boys, 17 girls), between 1 and 34 months of age (mean, 11.06 months), who underwent cranial expansion surgery for craniosynostosis affecting the coronal suture complex. Maxillary volumes were plotted against age for the first 3 years of life and were compared with a healthy population. There was no statistical difference between the two sexes for mean maxillary volume. The mean maxillary volumes for the entire group were statistically smaller than the norm (p = 0.046, linear regression with age as a covariable), but there was no statistical difference among the four different groups of coronal synostosis (unilateral coronal, nonsyndromic bilateral coronal, nonsyndromic complex pansynostosis, syndromic bilateral coronal synostosis) (p = 0.407, one-way analysis of variance). On graphic data analysis, the maxillary volume was smaller than the norm in craniosynostotic children who presented in the first few months of life. However, by 7 months of age in nonsyndromic bilateral coronal synostosis and by 17 months of age in syndromic bilateral coronal synostosis, the maxillary volumes had increased toward the norm. This implies that the effect of the craniosynostotic process on the midface structures is present from birth and parallels the effect on the cranial vault sutures.     

Evaluation of cranial bone suture autotransplants in the growing rabbit

A coronal bone suture segment was autotransplanted into an experimentally created defect in the nongrowing portion of the nasal bone in 12 5-week-old male New Zealand white rabbits. The animals were sacrificed 90 days postoperatively. In most specimens the transplants were well incorporated into the recipient site. The transplanted sutures appeared narrower radiographically, and the bony projections of the sutures were not as long as those seen in control sutures. In each case the transplanted sutural ligament had atrophied, and six of the transplants showed bony union across the sutural space. The results suggest that growth and biomechanical stresses are important for maintenance of the sutural ligament and for the stimulation of bone deposition.     

Increased IGF-I and IGF-II mRNA and IGF-I peptide in fusing rat cranial sutures suggest evidence for a paracrine role of insulin-like growth factors in suture fusion

Premature cranial suture fusion, or craniosynostosis, can result in gross aberrations of craniofacial growth. The biology underlying cranial suture fusion remains poorly understood. Previous studies of the Sprague-Dawley rat posterior frontal suture, which fuses at between 12 and 20 days, have suggested that the regional dura mater beneath the cranial suture directs the overlying suture's fusion. To address the dura-suture paracrine signaling that results in osteogenic differentiation and suture fusion, the authors investigated the possible role of insulin-like growth factors (IGF) I and II. The authors studied the temporal and spatial patterns of the expression of IGF-I and IGF-II mRNA and IGF-I peptide and osteocalcin (bone morphogenetic protein-4) protein in fusing posterior frontal rat sutures, and they compared them with patent coronal (control) sutures. Ten Sprague-Dawley rats were studied at the following time points: 16, 18, and 20 days of gestation and 2, 5, 10, 15, 20, 30, 50, and 80 days after birth (n = 110). Posterior frontal and coronal (patent, control) sutures were analyzed for IGF-I and IGF-II mRNA expression by in situ hybridization by using 35S-labeled IGF-I and IGF-II antisense riboprobes. Levels of IGF-I and IGF-II mRNA were quantified by counting the number of autoradiograph signals per cell. IGF-I and osteocalcin immunoreactivity were identified by avidin-biotin peroxidase immunohistochemistry. IGF-I and IGF-II mRNA were expressed in dural cells beneath fusing sutures, and the relative mRNA abundance increased between 2 and 10 days before initiation of fusion. Subsequently, IGF-I and IGF-II mRNA were detected in the suture connective tissue cells at 15 and 20 days during the time of active fusion. In contrast, within large osteoblasts of the osteogenic front, the expression of IGF-I and IGF-II mRNA was minimal. However, IGF-I peptide and osteocalcin protein were intensely immunoreactive within these osteoblasts at 15 days (during the period of suture fusion). These data suggest that the dura-suture interaction may be signaled in a paracrine fashion by dura-derived growth factors, such as IGF-I and IGF-II. These peptides, in turn, stimulate nearby osteoblasts to produce bone-promoting growth factors, such as osteocalcin.     

Craniofacial biology: Animal surgical experimentation and clinical practice

Studies are presented on the growth of the mandible in the pig, growth of the frontonasal suture and snout in the rabbit, and the development of the face and jaws in a human patient with anodontia. Growth of the snout after extirpation of the frontonasal suture is contrasted with its growth following resection of the cartilaginous nasal septum. The results of the studies have clinical applications in surgery and dentistry.     

Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-beta 3 activity and prevented by removal of TGF-beta 2 activity from fetal rat calvaria in vitro

Cranial suture morphogenesis requires soluble, heparin-binding factors secreted by the dura mater to resist premature osseous obliteration. Elevated levels of transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3 have previously been noted in cranial sutures undergoing normal and premature sutural obliteration. To examine the role of TGF-beta s in regulating cranial suture morphogenesis, an established in vitro, serum-free, calvarial culture system was used. In this system, fetal rat coronal sutures undergo apparently normal suture morphogenesis in the presence of dura mater, but undergo osseous obliteration in the absence of dura mater. Neutralizing polyclonal antibodies to TGF-beta 1, TGF-beta 2, or TGF-beta 3 were added to cultures of fetal day 19 rat calvaria, which were harvested at 3, 4, or 5 days, processed for histology, sectioned, and examined. Coronal sutures from calvaria cultured in the presence of dura mater resisted obliteration, either alone or in the presence of TGF-beta 1 or TGF-beta 2 neutralizing antibodies. However, sutures from calvaria cultured in the presence of TGF-beta 3 neutralizing antibodies became obliterated. Conversely, sutures from calvaria cultured in the absence of dura mater became obliterated by bone, either alone or in the presence of neutralizing antibodies to TGF-beta 1 or TGF-beta 3. However, those sutures cultured in the presence of neutralizing antibodies to TGF-beta 2 were rescued from osseous obliteration.     

The BMP ligand Gdf6 prevents differentiation of coronal suture mesenchyme in early cranial development

Growth Differentiation Factor-6 (Gdf6) is a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules. Previous studies have shown that Gdf6 plays a role in formation of a diverse subset of skeletal joints. In mice, loss of Gdf6 results in fusion of the coronal suture, the intramembranous joint that separates the frontal and parietal bones. Although the role of GDFs in the development of cartilaginous limb joints has been studied, limb joints are developmentally quite distinct from cranial sutures and how Gdf6 controls suture formation has remained unclear. In this study we show that coronal suture fusion in the Gdf6-/- mouse is due to accelerated differentiation of suture mesenchyme, prior to the onset of calvarial ossification. Gdf6 is expressed in the mouse frontal bone primordia from embryonic day (E) 10.5 through 12.5. In the Gdf6-/- embryo, the coronal suture fuses prematurely and concurrently with the initiation of osteogenesis in the cranial bones. Alkaline phosphatase (ALP) activity and Runx2 expression assays both showed that the suture width is reduced in Gdf6+/- embryos and is completely absent in Gdf6-/- embryos by E12.5. ALP activity is also increased in the suture mesenchyme of Gdf6+/- embryos compared to wild-type. This suggests Gdf6 delays differentiation of the mesenchyme occupying the suture, prior to the onset of ossification. Therefore, although BMPs are known to promote bone formation, Gdf6 plays an inhibitory role to prevent the osteogenic differentiation of the coronal suture mesenchyme.     

Intracranial pressure changes in craniosynostotic rabbits

Cranial vault and brain deformities in individuals with craniosynostosis are thought to result, in part, from changes in intracranial pressure, but clinical findings are still inconclusive. The present study describes intracranial pressure changes in a rabbit model with naturally occurring, uncorrected coronal suture synostosis. Longitudinal and cross-sectional intracranial pressure data were collected from 241 New Zealand White rabbits, divided into four groups: normal controls (n = 81); rabbits with delayed-onset coronal suture synostosis (n = 78); rabbits with early-onset unilateral coronal suture synostosis (n = 32); and rabbits with early-onset bilateral coronal suture synostosis (n = 50). Epidural intracranial pressure measurements were obtained at 10, 25, 42, and 84 days of age using a NeuroMonitor microsensor transducer. Normal rabbits and rabbits with delayed-onset coronal suture and early-onset unilateral coronal suture synostosis showed a similar oscillating pattern of age-related changes in normal and head-down intracranial pressure from 10 to 84 days of age. In contrast, rabbits with early-onset bilateral coronal suture synostosis showed markedly elevated normal and head-down intracranial pressure levels from 10 to 25 days and showed a different pattern through 84 days. Results from one-way analysis of variance revealed significant (p < 0.01) group differences only at 25 days of age. Rabbits with early-onset bilateral coronal suture synostosis had significantly (p < 0.05) greater normal and head-down intracranial pressure (by 42 percent) than the other three groups. These results showed differing intracranial pressure compensations in rabbits with uncorrected multiple-suture synostosis compared with normal rabbits or rabbits with uncorrected single-suture synostosis, possibly through progressive cerebral atrophy and decreased intracranial volume, abnormal intracranial vascular patterns and blood volume, and/or differing cranial vault compensatory changes.     

Experimental unilateral coronal synostosis in rabbits

Immobilization of the coronal suture was produced unilaterally in 9-day-old rabbits to determine its effect on subsequent craniofacial development. The suture was immobilized unilaterally by the topical application of methylcyanoacrylate adhesive. Subsequent growth effects on the cranial vault, base, and facial skeleton were assessed by serial radiographic cephalometry. Unilateral coronal suture immobilization resulted in significantly decreased bone growth at the coronal suture (mean 0.95 mm +/- 0.35 SE) when compared to sham-treated control animals (mean 5.06 mm +/- 0.20 SE). Frontonasal suture bone growth contralateral to the immobilized half of the coronal suture, however, was significantly increased. The anterior cranial base became significantly shortened, and orbital asymmetry developed. The pattern of induced abnormalities simulates unilateral coronal synostosis in humans.     

Tissue origins and interactions in the mammalian skull vault.

During mammalian evolution, expansion of the cerebral hemispheres was accompanied by expansion of the frontal and parietal bones of the skull vault and deployment of the coronal (fronto-parietal) and sagittal (parietal-parietal) sutures as major growth centres. Using a transgenic mouse with a permanent neural crest cell lineage marker, Wnt1-Cre/R26R, we show that both sutures are formed at a neural crest-mesoderm interface: the frontal bones are neural crest-derived and the parietal bones mesodermal, with a tongue of neural crest between the two parietal bones. By detailed analysis of neural crest migration pathways using X-gal staining, and mesodermal tracing by DiI labelling, we show that the neural crest-mesodermal tissue juxtaposition that later forms the coronal suture is established at E9.5 as the caudal boundary of the frontonasal mesenchyme. As the cerebral hemispheres expand, they extend caudally, passing beneath the neural crest-mesodermal interface within the dermis, carrying with them a layer of neural crest cells that forms their meningeal covering. Exposure of embryos to retinoic acid at E10.0 reduces this meningeal neural crest and inhibits parietal ossification, suggesting that intramembranous ossification of this mesodermal bone requires interaction with neural crest-derived meninges, whereas ossification of the neural crest-derived frontal bone is autonomous. These observations provide new perspectives on skull evolution and on human genetic abnormalities of skull growth and ossification.     

Strain in the braincase and its sutures during function

The skull is distinguished from other parts of the skeleton by its composite construction. The sutures between bony elements provide for interstitial growth of the cranium, but at the same time they alter the transmission of stress and strain through the skull. Strain gages were bonded to the frontal and parietal bones of miniature pigs and across the interfrontal, interparietal and coronal sutures. Strains were recorded 1) during natural mastication in conjunction with electromyographic activity from the jaw muscles and 2) during stimulation of various cranial muscles in anesthetized animals. Vault sutures exhibited vastly higher strains than did the adjoining bones. Further, bone strain primarily reflected torsion of the braincase set up by asymmetrical muscle contraction; the tensile axis alternated between +45 degrees and -45 degrees depending on which diagonal masseter/temporalis pair was most active. However, suture strains were not related to overall torsion but instead were responses to local muscle actions. Only the coronal suture showed significant strain (tension) during jaw opening; this was caused by the contraction of neck muscles. All sutures showed strain during jaw closing, but polarity depended on the pattern of muscle usage. For example, masseter contraction tensed the coronal suture and the anterior part of the interfrontal suture, whereas the temporalis caused compression in these locations. Peak tensile strains were larger than peak compressive strains. Histology suggested that the skull is bent at the sutures, with the ectocranial surface tensed and the endocranial surface predominantly compressed. Collectively, these results indicate that skulls with patent sutures should be analyzed as complexes of independent parts rather than solid structures.     

Growth of calvarial width. An experimental investigation in rabbits

This investigation was conducted to analyze growth in width of the rabbit calvarium. 15 male New Zealand white rabbits were subjected to regular stereometric examinations from 31 to 141 days of age after implantation of tantalum bone markers. The sagittal suture complex, i.e. the interfrontal and interparietal sutures, and the bilateral temporal sutures demonstrated similar growth rates in magnitude which moderately decelerated throughout the observation period. Transverse growth exhibited local growth fluctuations and short-term negative growth values in a well-balanced manner.     

Craniosynostosis and altered patterns of fetal TGF-beta expression induced by intrauterine constraint

Recent work has demonstrated that fusion of the calvarial sutures is mediated by locally elaborated soluble growth factors, including the transforming growth factor-betas (TGF-betas), leading some to speculate that external biomechanical forces play little role in suture development. Clinical evidence has long suggested, however, that fetal head constraint may play a critical role in the pathogenesis of many cases of nonsyndromic craniosynostosis. The purpose of these experiments was to test the hypothesis that intrauterine constraint leads to an alteration in normal patterns of TGF-beta expression and that these alterations are associated with craniosynostosis. Fetal constraint was induced by allowing C57Bl/6 murine fetuses to grow for 2.5 days beyond the normal 20-day gestation by performing uterine cerclage on the eighteenth day. Cranial suture morphology was examined in hematoxylin and eosin-stained sections and in cleared whole-mount specimens, double stained with alizarin red S and Alcian blue. Expression patterns of TGF-beta1 and TGF-beta3 were examined by immunohistochemical techniques. Gross and microscopic examination of the cranial sutures of 17 constrained fetuses revealed changes that ranged from narrowing to complete osseous obliteration of the coronal and squamosal sutures. All sutures of 14 nonconstrained control pups remained patent. Fetal head constraint was associated with increased TGF-beta1 immunoreactivity within the new bone and the underlying dura when compared with nonconstrained age-matched controls. TGF-beta3 immunoreactivity was associated with the dura underlying patent, nonconstrained sutures, whereas constraint-induced synostosis was characterized by down-regulation of dural TGF-beta3 expression. These experiments confirm the ability of intrauterine constraint to induce premature fusion of the cranial sutures and provide evidence that intrauterine head constraint induces the expression of osteogenic growth factors in fetal calvarial bone and the underlying dura.