Restricted growth at the frontonasal suture: alterations in craniofacial growth in rabbits

Apposition of bone at the sutural margin is generally thought to be a compensatory adjustment to growing soft-tissue organs such as the brain or eyes within the skull. The frontonasal suture which is located at the interface between the cranial and facial skeletons is a site of extremely active growth in the young rabbit. Recently, we showed that premature closure of a cranial suture, the coronal suture, can alter the growth not only at the adjacent frontonasal suture but also of the basicranium and midface. This study examines the effects of restricted growth at the frontonasal suture on both growth at adjacent cranial sutures and linear growth of the basicranium and midface. Thirty newborn New Zealand White rabbits were subdivided into experimental and sham-treated groups of equal size and distribution for sex and birth weight. At 9 days of age, the frontonasal suture of each experimental animal was immobilized by bilateral application of methyl-cyanoacrylate adhesive across the frontonasal suture. Growth and morphometric changes were monitored by radiocephalometric methods through 120 days of age by bilateral implantation of radiopague markers on each side of frontonasal, coronal, and anterior lambdoid sutures. Results indicate that restricted growth at the frontonasal suture results not only in a significant shortening of the midface but also in significant decreases in growth at the coronal and internasal sutures. Growth at the interfrontal and sagittal sutures is increased. Furthermore, growth at the anterior portion of the nasal bones is significantly increased, thereby offsetting a portion of the decreased nasal bone length resulting from frontonasal restriction.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Timing of Egf treatment differentially affects Tgf-beta2 induced cranial suture closure

Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-beta 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-beta 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-beta 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-beta 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-beta 2-induced suture closure when Egf was given either prior to or after Tgf-beta 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-beta 2. However, Egf added after Tgf-beta 2 treatment had no effect on Tgf-beta 2-induced suture closure. Blocking Egf activity after Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced obliteration, indicating that Tgf-beta 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-beta 2 is critical for normal suture development and maintenance of suture patency.     

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.     

Size and shape of human cranial sutures--a new scoring method

A method for the differentiation of sutural patterns of the human cranial vault is introduced. Three criteria of differentiation are considered, one for size and two for shape: 1) maximal shape extension; 2) basic configuration; 3) secondary protrusion. The method is illustrated here for the coronal and lambdoid sutures of 70 recent Italian skulls (35 adult males and 35 adult females). Differences between coronal and lambdoid sutural size and shape can be detected analytically; for example, the coronal suture commonly shows lesser degrees of shape extension, a simpler basic configuration, and an absence of secondary protrusion. Heterogeneity within each suture, as well as a relationship among corresponding sections and between the three criteria adopted, have been also observed; symmetry predominates for both the sutures, and sexual differences are slight.     

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.     

Fusion patterns of craniofacial sutures in rhesus monkey skulls of known age and sex from Cayo Santiago

Bones of the face and cranial vault meet at sutural boundaries. These sutures are of great importance for craniofacial growth. Although the effects that the sutures have on modulating craniofacial strains have been investigated, how sutural fusion influences primate craniofacial biomechanics and adaptation are less considered. Confounding this problem is the lack of any systematic data on patterns of craniofacial sutural fusion from animals of known age and sex. This study examined the status of 28 sutures in Macaca mulatta skulls from a collection of animals of known age and sex from Cayo Santiago, Puerto Rico. Survival analysis showed that most animals died before all sutures fused. There was high variation in the age at which individual sutures or sutural sections were fused in M. mulatta, and significant differences in the amount of sutural fusion among regions and between males and females. Intensive fusion of sutures took place between ages 5 and 15. Sutures in the facial area tended to be less fused than in the cranial vault. Between adolescence and adulthood, males tended to have more sutural fusion than females, especially in the facial area. These differences might be biomechanical adaptations during ontogeny to craniofacial sexual dimorphism. These findings enrich our understanding of variation in sutural morphology in rhesus monkeys. Comparative information across primate species is essential for understanding the biomechanics of craniofacial form throughout primate evolution.     

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.     

BMP9 induces osteogenesis and adipogenesis in the immortalized human cranial suture progenitors from the patent sutures of craniosynostosis patients

The cranial suture complex is a heterogeneous tissue consisting of osteogenic progenitor cells and mesenchymal stem cells (MSCs) from bone marrow and suture mesenchyme. The fusion of cranial sutures is a highly coordinated and tightly regulated process during development. Craniosynostosis is a congenital malformation caused by premature fusion of cranial sutures. While the progenitor cells derived from the cranial suture complex should prove valuable for studying the molecular mechanisms underlying suture development and pathogenic premature suture fusion, primary human cranial suture progenitors (SuPs) have limited life span and gradually lose osteoblastic ability over passages. To overcome technical challenges in maintaining sufficient and long‐term culture of SuPs for suture biology studies, we establish and characterize the reversibly immortalized human cranial suture progenitors (iSuPs). Using a reversible immortalization system expressing SV40 T flanked with FRT sites, we demonstrate that primary human suture progenitor cells derived from the patent sutures of craniosynostosis patients can be efficiently immortalized. The iSuPs maintain long‐term proliferative activity, express most of the consensus MSC markers and can differentiate into osteogenic and adipogenic lineages upon BMP9 stimulation in vitro and in vivo. The removal of SV40 T antigen by FLP recombinase results in a decrease in cell proliferation and an increase in the endogenous osteogenic and adipogenic capability in the iSuPs. Therefore, the iSuPs should be a valuable resource to study suture development, intramembranous ossification and the pathogenesis of craniosynostosis, as well as to explore cranial bone tissue engineering.     

Differential activation of canonical Wnt signaling determines cranial sutures fate: A novel mechanism for sagittal suture craniosynostosis

Premature closure of cranial sutures, which serve as growth centers for the skull vault, result in craniosynostosis. In the mouse posterior frontal (PF) suture closes by endochondral ossification, whereas sagittal (SAG) remain patent life time, although both are neural crest tissue derived. We therefore, investigated why cranial sutures of same tissue origin adopt a different fate. We demonstrated that closure of the PF suture is tightly regulated by canonical Wnt signaling, whereas patency of the SAG suture is achieved by constantly activated canonical Wnt signaling. Importantly, the fate of PF and SAG sutures can be reversed by manipulating Wnt signaling. Continuous activation of canonical Wnt signaling in the PF suture inhibits endochondral ossification and therefore, suture closure, In contrast, inhibition of canonical Wnt signaling in the SAG suture, upon treatment with Wnt antagonists results in endochondral ossification and suture closure. Thus, inhibition of canonical Wnt signaling in the SAG suture phenocopies craniosynostosis. Moreover, mice haploinsufficient for Twist1, a target gene of canonical Wnt signaling which inhibits chondrogenesis, have sagittal craniosynostosis. We propose that regulation of canonical Wnt signaling is of crucial importance during the physiological patterning of PF and SAG sutures. Importantly, dysregulation of this pathway may lead to craniosynostosis.