Positional changes of the frontoparietal ossification centers in perinatal craniosynostotic rabbits.

It has been suggested that craniosynostosis is caused by abnormally located ossification centers (i.e., bony tubers) in the developing skull prior to suture formation [Mathijssen et al., 1996, 1997]. The present study was designed to test this hypothesis in a rabbit model of human familial, nonsyndromic coronal suture (CS) synostosis. Calvariae were taken from 99 New Zealand White rabbit perinates (55 normal controls, 15 with delayed-onset CS synostosis, and 29 with bilateral or unilateral CS synostosis), ranging in age from 23 to 34 days postconception (synostosis occurs at approximately 23 days in this model). Frontoparietal, interfrontal, and interparietal ossification center distances were obtained using a Wild microscope with camera lucida attachment and a 2-D computer digitization technique. Linear regression analysis was used to compare age-related changes in the perinatal ossification centers among groups. Results revealed that frontoparietal ossification center regression line slopes had similar start points (24-day intercepts) with significantly (P < 0.05) diverging slopes over time. Normal and delayed-onset ossification center distance increased more rapidly than in synostosed perinates. No significant (P > 0.05) differences were noted in regression line slopes among groups for interparietal or interfrontal ossification center distances. Results demonstrated that, in synostosed perinates, frontoparietal ossification center location was similar to normals around the time of synostosis and became displaced later. These findings suggest that ossification center (i.e., bony tuber) displacement seen in infants with craniosynostosis is probably a secondary and compensatory, postsynostotic change and not a primary causal factor of synostosis in this rabbit model.     

Metopic synostosis: Defining the temporal sequence of normal suture fusion and differentiating it from synostosis on the basis of computed tomography images

Only the metopic suture normally fuses during early childhood; all other cranial sutures normally fuse much later in life. Despite this, metopic synostosis is one of the least common forms of craniosynostosis. The temporal sequence of normal physiologic metopic suture fusion remains undefined and controversial. Therefore, diagnosis of metopic synostosis on the basis of computed tomography images alone can prove misleading. The present study sought to determine the normal sequence of metopic suture fusion and characterize both endocranial and ectocranial suture morphology. An analysis of computed tomography scans of 76 trauma patients, ranging in age from 10 days to 18 months, provided normative craniofacial data that could be compared to similar data obtained from the preoperative computed tomography scans of 30 patients who had undergone surgical treatment for metopic synostosis. Metopic suture fusion was complete by 6 to 8 months in all nonsynostotic patients, with initiation of suture fusion evident as early as 3 months of age. Fusion was found to commence at the nasion, proceed superiorly in progressive fashion, and conclude at the anterior fontanelle. Although an endocranial ridge was not commonly seen in synostotic patients, an endocranial metopic notch was virtually diagnostic of premature suture fusion and was seen in 93 percent of synostotic patients. A metopic notch was not seen in any nonsynostotic patient. The morphologic and normative craniofacial data presented permit diagnosis of metopic synostosis based on computed tomography images obtained beyond the normal fusion period.     

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.     

Simultaneous induction of apoptosis, collagen type I expression and mineralization in the developing coronal suture following FGF4 and FGF2 application

This study aimed to evaluate the disturbances in normal coronal suture development resulting in craniosynostosis, a congenital disorder in which the calvarial sutures close prematurely. Craniosynostosis syndromes can be caused by mutations in the genes encoding for the fibroblast growth factor receptors (FGFRs) 1, 2, and 3. These gain-of-function mutations cause the transcribed receptor to be constitutively activated. To mimic this genetic defect, fibroblast growth factor (FGF) 2 or 4 was administered near the developing coronal suture in normal mouse embryos through ex utero surgery. The effect on apoptosis and bone differentiation, as collagen type I expression and mineralization, within the FGF-exposed coronal suture was investigated through (immuno)histochemical staining. An increase in the number of apoptotic cells together with ectopic collagen type I expression within the suture and accelerated mineralization followed FGF application. Macroscopically, this presented as a synostotic coronal suture. These results suggest that both apoptosis and differentiation are two processes that are simultaneously implicated in synostosis of the coronal suture in case of a FGFR-related craniosynostosis.     

Sustained Platelet-Derived Growth Factor Receptor α Signaling in Osteoblasts Results in Craniosynostosis by Overactivating the Phospholipase C-γ Pathway

The development and growth of the skull is controlled by cranial sutures, which serve as growth centers for osteogenesis by providing a pool of osteoprogenitors. These osteoprogenitors undergo intramembranous ossification by direct differentiation into osteoblasts, which synthesize the components of the extracellular bone matrix. A dysregulation of osteoblast differentiation can lead to premature fusion of sutures, resulting in an abnormal skull shape, a disease called craniosynostosis. Although several genes could be linked to craniosynostosis, the mechanisms regulating cranial suture development remain largely elusive. We have established transgenic mice conditionally expressing an autoactivated platelet-derived growth factor receptor α (PDGFRα) in neural crest cells (NCCs) and their derivatives. In these mice, premature fusion of NCC-derived sutures occurred at early postnatal stages. In vivo and in vitro experiments demonstrated enhanced proliferation of osteoprogenitors and accelerated ossification of osteoblasts. Furthermore, in osteoblasts expressing the autoactivated receptor, we detected an upregulation of the phospholipase C-γ (PLC-γ) pathway. Treatment of differentiating osteoblasts with a PLC-γ-specific inhibitor prevented the mineralization of synthesized bone matrix. Thus, we show for the first time that PDGFRα signaling stimulates osteogenesis of NCC-derived osteoblasts by activating the PLC-γ pathway, suggesting an involvement of this pathway in the etiology of human craniosynostosis.     

Calvarial sutural abnormalities: metopic synostosis and coronal deformation--an anatomic, three-dimensional radiographic, and pathologic study

The purposes of this study were (1) to evaluate the histologic differences between synostotic versus deformational suture abnormalities and (2) to correlate these histologic findings with anatomic and three-dimensional computed tomographic (CT) scans. We examined three infants with premature metopic synostosis; one infant also had microcephaly trisomy 13 and curious overriding of the coronal sutures. The three-dimensional CT scans demonstrated obliteration of the metopic suture inferiorly. Histologic sections of this suture showed complete bony stenosis. The same pattern was found in all three infants, including the two infants with trigonocephaly who did not have trisomy 13 or microcephaly. In the trisomy 13 infant, the overlapped inferior coronal suture was obliterated on CT examination. However, histologic sections in this region showed a merging of bone; there was no synostosis. In summary, three-dimensional CT re-formation correlated with metopic suture histology. "Stenotic" fusion existed in all infants with trigonocephaly, those with normal and abnormal karyotypes, with and without microcephaly. However, three-dimensional CT re-formation of the trisomic infant showed opacification of the coronal suture in the areas of greatest overlap, whereas histology revealed a curious bone remodeling pattern, possibly a precursor to "deformational" craniosynostosis.     

Mesodermal expression of Fgfr2S252W is necessary and sufficient to induce craniosynostosis in a mouse model of Apert syndrome

Coordinated growth of the skull and brain are vital to normal human development. Craniosynostosis, the premature fusion of the calvarial bones of the skull, is a relatively common pediatric disease, occurring in 1 in 2500 births, and requires significant surgical management, especially in syndromic cases. Syndromic craniosynostosis is caused by a variety of genetic lesions, most commonly by activating mutations of FGFRs 1-3, and inactivating mutations of TWIST1. In a mouse model of TWIST1 haploinsufficiency, cell mixing between the neural crest-derived frontal bone and mesoderm-derived parietal bone accompanies coronal suture fusion during embryonic development. However, the relevance of lineage mixing in craniosynostosis induced by activating FGFR mutations is unknown. Here, we demonstrate a novel mechanism of suture fusion in the Apert Fgfr2(S252W) mouse model. Using Cre/lox recombination we simultaneously induce expression of Fgfr2(S252W) and β-galactosidase in either the neural crest or mesoderm of the skull. We show that mutation of the mesoderm alone is necessary and sufficient to cause craniosynostosis, while mutation of the neural crest is neither. The lineage border is not disrupted by aberrant cell migration during fusion. Instead, the suture mesenchyme itself remains intact and is induced to undergo osteogenesis. We eliminate postulated roles for dura mater or skull base changes in craniosynostosis. The viability of conditionally mutant mice also allows post-natal assessment of other aspects of Apert syndrome.     

Craniosynostosis and multiple skeletal anomalies in humans and zebrafish result from a defect in the localized degradation of retinoic acid

Excess exogenous retinoic acid (RA) has been well documented to have teratogenic effects in the limb and craniofacial skeleton. Malformations that have been observed in this context include craniosynostosis, a common developmental defect of the skull that occurs in 1 in 2500 individuals and results from premature fusion of the cranial sutures. Despite these observations, a physiological role for RA during suture formation has not been demonstrated. Here, we present evidence that genetically based alterations in RA signaling interfere with human development. We have identified human null and hypomorphic mutations in the gene encoding the RA-degrading enzyme CYP26B1 that lead to skeletal and craniofacial anomalies, including fusions of long bones, calvarial bone hypoplasia, and craniosynostosis. Analyses of murine embryos exposed to a chemical inhibitor of Cyp26 enzymes and zebrafish lines with mutations in cyp26b1 suggest that the endochondral bone fusions are due to unrestricted chondrogenesis at the presumptive sites of joint formation within cartilaginous templates, whereas craniosynostosis is induced by a defect in osteoblastic differentiation. Ultrastructural analysis, in situ expression studies, and in vitro quantitative RT-PCR experiments of cellular markers of osseous differentiation indicate that the most likely cause for these phenomena is aberrant osteoblast-osteocyte transitioning. This work reveals a physiological role for RA in partitioning skeletal elements and in the maintenance of cranial suture patency.     

Neurodevelopment in children with single-suture craniosynostosis and plagiocephaly without synostosis.

The objective of this study was to determine whether children with nonsyndromic craniosynostosis and plagiocephaly without synostosis demonstrated cognitive and psychomotor delays when compared with a standardized population sample. This was the initial assessment of a larger prospective study, which involved 21 subjects with nonsyndromic craniosynostosis (mean age, 10.9 months) and 42 subjects with plagiocephaly without synostosis (mean age, 8.4 months). Each child was assessed using the Bayley Scales of Infant Development-II (BSID-II) for cognitive and psychomotor development before therapeutic intervention (surgery for craniosynostosis and molding-helmet therapy for plagiocephaly without synostosis). The distribution of the scores was divided into four groups: accelerated, normal, mild delay, and significant delay. The distributions of the mental developmental index (MDI) and the psychomotor developmental index (PDI) were then compared with a standardized Bayley's age-matched population, using Fisher's exact chi-square test. Within the craniosynostosis group, the PDI scores were significantly different from the standardized distribution (p < 0.001). With regard to the PDI scores, 0 percent of the subjects in the craniosynostosis group were accelerated, 43 percent were normal, 48 percent had mild delay, and 9 percent had significant delay. In contrast, the MDI scores were not statistically different (p = 0.08). Within the group with plagiocephaly without synostosis, both the PDI and MDI scores were significantly different from the normal curve distribution (p < 0.001). With regard to the PDI scores, 0 percent of the subjects in the group with plagiocephaly without synostosis were accelerated, 67 percent were normal, 20 percent had mild delay, and 13 percent had significant delay. With regard to the MDI scores, 0 percent of the subjects in this group were accelerated, 83 percent were normal, 8 percent had mild delay and 9 percent had significant delay. This study indicates that before any intervention, subjects with single-suture syndromic craniosynostosis and plagiocephaly without synostosis demonstrate delays in cognitive and psychomotor development. Continued postintervention assessments are needed to determine whether these developmental delays can be ameliorated with treatment.     

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.     

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.     

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.     

Unilateral fusion of the frontosphenoidal suture: a rare cause of synostotic frontal plagiocephaly

Unilateral coronal synostosis is the common appellation for premature, one-sided fusion of the frontoparietal suture-the most common cause of synostotic frontal plagiocephaly. However, frontal asymmetry can also result from isolated fusion across the anterior cranial base without involvement of the frontoparietal suture. This article describes three patients with localized synostosis of the frontosphenoidal suture, the medial extension of the coronal ring. Two patients were initially misdiagnosed as having unilateral coronal synostosis and the other as having deformational frontal plagiocephaly. The patients had variable frontal flattening, with depression and recession of the ipsilateral orbital rim. The nasal root was midline or slightly deviated to the contralateral side. The sagittal position of the ipsilateral malar eminence was slightly retruded in one patient and symmetric in the other two. The auricular position was symmetric in the sagittal plane for all patients. In all three patients, computed tomography examination demonstrated a patent frontoparietal suture and fusion of the frontosphenoidal suture (basilar hemicoronal ring). Two patients had involvement of contiguous sutures: one had fusion extending to the sphenoethmoidal suture and the other's involved part of the sphenozygomatic suture. The sagittal suture was midline in all patients. In summary, synostotic frontal plagiocephaly denotes a relatively broad phenotypic spectrum that includes unilateral coronal synostosis and more isolated fusions in the basilar coronal ring. The physical findings resulting from frontosphenoidal synostosis are unique, yet careful evaluation will minimize confusion with other causes of asymmetric frontal flattening. Proper diagnosis necessitates awareness of this uncommon entity and requires focused computed tomographic assessment.     

A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plastiCity among diplomonads and significant lateral gene transfer in eukaryote genome evolution

Background

Craniosynostosis, the premature fusion of calvarial sutures, is a common craniofacial abnormality. Causative mutations in more than 10 genes have been identified, involving fibroblast growth factor, transforming growth factor beta, and Eph/ephrin signalling pathways. Mutations affect each human calvarial suture (coronal, sagittal, metopic, and lambdoid) differently, suggesting different gene expression patterns exist in each human suture. To better understand the molecular control of human suture morphogenesis we used microarray analysis to identify genes differentially expressed during suture fusion in children with craniosynostosis. Expression differences were also analysed between each unfused suture type, between sutures from syndromic and non-syndromic craniosynostosis patients, and between unfused sutures from individuals with and without craniosynostosis.

Results

We identified genes with increased expression in unfused sutures compared to fusing/fused sutures that may be pivotal to the maintenance of suture patency or in controlling early osteoblast differentiation (i.e. RBP4, GPC3, C1QTNF3, IL11RA, PTN, POSTN). In addition, we have identified genes with increased expression in fusing/fused suture tissue that we suggest could have a role in premature suture fusion (i.e. WIF1, ANXA3, CYFIP2). Proteins of two of these genes, glypican 3 and retinol binding protein 4, were investigated by immunohistochemistry and localised to the suture mesenchyme and osteogenic fronts of developing human calvaria, respectively, suggesting novel roles for these proteins in the maintenance of suture patency or in controlling early osteoblast differentiation. We show that there is limited difference in whole genome expression between sutures isolated from patients with syndromic and non-syndromic craniosynostosis and confirmed this by quantitative RT-PCR. Furthermore, distinct expression profiles for each unfused suture type were noted, with the metopic suture being most disparate. Finally, although calvarial bones are generally thought to grow without a cartilage precursor, we show histologically and by identification of cartilage-specific gene expression that cartilage may be involved in the morphogenesis of lambdoid and posterior sagittal sutures.

Conclusion

This study has provided further insight into the complex signalling network which controls human calvarial suture morphogenesis and craniosynostosis. Identified genes are candidates for targeted therapeutic development and to screen for craniosynostosis-causing mutations.     

Management of craniosynostosis

Learning Objectives: After studying this article, the participant should be able to: 1. Review the etiopathogenesis of craniosynostosis and craniofacial anomalies. 2. Develop a basic understanding of the clinical manifestations and diagnosis of craniofacial anomalies. 3. Describe the surgical principles of managing craniosynostosis and craniofacial anomalies.Craniosynostosis, or the premature closure of calvarial sutures, results in deformed calvaria at birth. Although the etiology of craniosynostosis is currently unknown, animal experiments and a recent interest in molecular biology point toward interplay between the dura and the underlying brain. This interaction occurs by means of a local alteration in the expression of transforming growth factor, MSX2, fibroblast growth factor receptor, and TWIST. The fused suture restricts growth of the calvaria, thus leading to a characteristic deformation, each associated with a different type of craniosynostosis. Uncorrected craniosynostosis leads to a continuing progression of the deformity, and in some cases, an elevation of intracranial pressure. Clinical examination should include not only an examination of the skull but also a general examination to rule out the craniofacial syndromes that accompany craniosynostosis. Because deformational plagiocephaly, or plagiocephaly without synostosis, occurs secondary to sleeping in the supine position during the early perinatal period, the physician should be aware of this abnormality. Treatment for deformational plagiocephaly is conservative when compared with treatment for craniosynostosis, which requires surgery. Appropriate investigations should include genetic screening, radiologic examination with a computerized tomographic scan, and neurodevelopmental analysis. Surgical intervention should be performed during infancy, preferably in the first 6 months of postnatal life, to prevent the further progression of the deformity and possible complications associated with increased intracranial pressure. The principles of surgical intervention are not only to excise the fused suture but also to attempt to normalize the calvarial shape. Long-term follow-up is critical to determine the effect of the surgical outcome.     

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.     

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.