Osteogenic Fronts Research Articles

Correction of Coronal Suture Synostosis Using Suture and Dura Mater Allografts in Rabbits With Familial Craniosynostosis

Abstract Objective: Resynostosis following surgical correction of craniosynostosis is a common clinical correlate. Recent studies suggest that the dura mater is necessary to maintain suture patency. It has also been hypothesized that dura mater from synostotic individuals may provide aberrant biochemical signals to the osteogenic fronts of the calvaria, which result in premature suture fusion and subsequent resynostosis following surgery. This study was designed to test this hypothesis by surgically manipulating the coronal suture and dura mater in rabbits with familial craniosynostosis to prevent postsurgical resynostosis. Design: Craniofacial growth and histomorphometric data were collected from 129 rabbits: 72 normal controls and 57 rabbits with bilateral coronal suture synostosis (15 unoperated on controls; 13 surgical controls; 9 dura mater transplant only; 10 suture transplant only; and 10 suture and dura mater transplant). At 10 days of age, all rabbits had radiopaque amalgam markers placed on eith...

A TWIST in the fate of human osteoblasts identifies signaling molecules involved in skull development.

The twist gene was first recognized by the Nobel Prize–winning laboratory of Nusslein-Volhard (1) and described in Drosophila as a zygotic gene required for dorsoventral patterning (2). Along the dorsoventral axis during the blastoderm stages of Drosophila, the most ventral nuclei constitute the mesodermal anlage which gives rise to cells that invaginate at gastrulation. With the loss of twist expression, homozygous mutant embryos fail to develop a mesoderm layer, resulting in few if any mesodermally derived internal organs. The head segments appear everted due to abnormal head involution, and the anterior end of the embryo is “twisted” in the egg. In mice, Twist expression also is initially observed along the dorsoventral gradient and then in the mesoderm and neural crest cell derivatives (3). Later, Twist is found in the somites, head mesenchyme, aortic arches, branchial arches, limb buds, and mesenchyme underneath the epidermis (4). Twist is expressed in the undifferentiated cells committed to muscle and cartilage development and inhibits their differentiation. Its transcripts are present in primary osteoblastic cells of the newborn mouse calvaria (5). Mouse embryos homozygous for the disruption of the Twist gene exhibit defects in the head mesenchyme, failure of neural tube closure in the cranial region, and pyknotic nuclei found in cranial nerves (6). Heterozygous mice display skull defects of poorly developed squamosal bones and of overdeveloped interparietal bones and limb abnormalities (7). The conservation of the Twist mutant phenotype also extends to humans. The heterozygous loss of the TWIST gene or its function results in a common autosomal dominant syndrome, Saethre-Chotzen, which is characterized by craniosynostosis, facial dysmorphisms, and hand and foot abnormalities (8, 9). Of these developmental abnormalities, craniosynostosis — the premature fusion of cranial sutures — is the most notable. Among the cranial sutures, the coronal suture across the width of the skull is most often prematurely synostosed, leading to a brachycephalic skull — one that is box-shaped and of shortened length. Mineralization of the cranium normally occurs directly from membrane-derived paraxial mesoderm. Ossification centers coalesce to form bones, and their osteogenic fronts meet to induce sutures along their lines of approximation. The skull continues to grow by appositional growth at the suture, with deposition of premineralized bone matrix along the suture margins. Several hypotheses have been proposed that premature synostosis is due to the abnormal development of the mesodermal blastema, accelerated bone growth, and/or increased differentiation. The discovery of the new disease gene TWIST generates an important tool that potentially could help in elucidating the signaling pathway and pathogenesis involved in craniosynostosis. A study reported in this issue of the JCI is evidence that this potential is being realized. Yousfi and colleagues from the Marie laboratory (10) identified TWIST target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome. This individual carries a previously reported and apparently recurrent point mutation in TWIST that introduces a premature stop codon (8, 9, 11). The product of this Y103X mutant allele would be predicted to be truncated upstream of the basic helix-loop-helix (bHLH) domain, which is normally required for dimerization and for DNA binding. However, since the patient’s osteoblasts showed decreased TWIST mRNA and protein levels, it seems likely that mRNAs carrying this mutation do not survive nonsense-mediated decay and that the Y103X allele is a true null. In vitro and in vivo experiments with TWIST mutant cells showed increased cell growth and ability to form bonelike nodular structures. These cells and aggregates exhibited increased expression of osteoblastic markers, alkaline phosphatase, and type I collagen, independent of cell growth. However, TWIST mutant cells showed reduction of osteocalcin mRNA expression during osteogenesis. The altered expression of these osteoblastic genes with craniosynostosis is consistent with previously published observations (5, 12, 13). Overexpression of TWIST by transfection studies in osteosarcoma cells may inhibit osteoblast differentiation (12), and reduction of murine Twist expression occurs with osteoblastic maturation (5). Osteocalcin-deficient mice have increased bone formation (13). Therefore, the significance of the findings from the Marie laboratory (10) is that the premature ossification of cranial sutures in Saethre-Chotzen syndrome patients with TWIST mutations is caused by increased osteoblastic cell proliferation and differentiation (Figure ​(Figure11). Figure 1 Elements involved in the molecular pathway of sutural osteoblast differentiation (modified from ref. 15). Top: normal cellular differentiation and relationship of TWIST and FGFR signaling. Bottom: altered gene expression in TWIST Y103X mutant osteoblasts ...

Individual osteoblasts in the developing calvaria express different gene repertoires

Several studies in vitro and a few in vivo have suggested that mature osteoblasts heterogeneously express osteoblast markers. In one recent study of the osteoblasts associated with bone nodules formed in vitro in rat calvaria cell populations, extensive diversity was documented in the overall gene repertoires expressed. To address whether comparable heterogeneity is evident in vivo, we investigated the expression of nine osteoblast lineage markers by both in situ hybridization and immunohistochemistry. At 21 days of fetal rat development, the calvaria is a rapidly growing bone with distinct maturational zones that are readily observed in coronal sections; that is, an osteogenic front emerging at sagittal and coronal sutures is adjacent to areas of growing trabeculae of bone, followed by more mature areas of remodeling bone. Based on expression patterns, markers can be divided into two categories. One category comprises markers that are globally expressed by all osteoblasts irrespective of their position in the calvaria. Of those tested, only two, alkaline phosphatase and the pth/pthrp receptor, fit into this category. All other markers analyzed, including transcription factors ( c-fos and msx-2), matrix molecules ( bone sialoprotein, osteopontin, and osteocalcin), and a hormone ( pthrp), were differentially expressed only in subpopulations of osteoblasts, based on cell maturational status, environment (ectocranial vs. endocranial surfaces), and microenvironment (adjacent osteoblasts). Preosteoblasts and osteocytes in different regions of the calvaria also expressed different subsets of the lineage markers. Mechanisms responsible for generating differential gene expression profiles appear to be both transcriptional and posttranscriptional. These results indicate that postproliferative, morphologically indistinguishable osteoblasts are not a homogeneous class of cells, but instead are molecularly diverse. The present results also raise the possibility that lineage progression and/or maintenance of the differentiated state may be adaptable in the calvaria.

Advances In Murine Cranial Suture Research

Craniosynostosis is a pathologic condition that results from premature fusion of one or more cranial sutures. It occurs in approximately 1:2000 live births. Since the brain expands rapidly in the first few years of life, premature closure of a cranial suture leads to compensatory calvarial expansion in a plane parallel to the fused suture. Untreated, craniosynostosis can cause a characteristic dysmorphic calvarial shape, midface hypoplasia, and can lead to deafness, blindness and mental retardation. In order to understand the dynamic mechanisms that mediate craniosynostosis, we needed to investigate the biologic processes, before, during and after suture fusion. Since clinical specimens limit our investigation to the time at which the samples are excised, we have employed murine models to examine the cascading events that lead to cranial suture fusion. These models have enabled us to dissect, isolate and understand the individual roles of the dura mater, pericranium, suture mesenchyme and osteogenic fronts.

The pathogenesis of craniosynostosis in the fetus.

Craniosynostosis occurs in approximately 1:2000 live births. It may affect the coronal, sagittal, metopic and lambdoid sutures in isolation or in combination. Although non-syndromic synostoses are more common, over 150 genetic syndromes have been identified. Recent advances in genetic mapping have linked chromosomal mutations with craniosynostotic syndromes. Despite the identification of these genetic mutations, the fundamental biomolecular mechanisms mediating cranial suture biology remain unknown. Today, many laboratories are investigating murine cranial suture biology as a model for human cranial suture development and fusion. Normal murine cranial suture biology is very complex, but evidence suggests that the dura mater provides the biomolecular blueprints (e.g. the soluble growth factors), which guide the fate of the pleuripotent osteogenic fronts. While our knowledge of these dura-derived signals has increased dramatically in the last decade, we have barely begun to understand the fundamental mechanisms that mediate cranial suture fusion or patency. Interestingly, recent advances in both premature human and programmed murine suture fusion have revealed unexpected results, and have generated more questions than answers.

Advances In Murine Cranial Suture Research

Craniosynostosis is a pathologic condition that results from premature fusion of one or more cranial sutures. It occurs in approximately 1:2000 live births. Since the brain expands rapidly in the first few years of life, premature closure of a cranial suture leads to compensatory calvarial expansion in a plane parallel to the fused suture. Untreated, craniosynostosis can cause a characteristic dysmorphic calvarial shape, midface hypoplasia, and can lead to deafness, blindness and mental retardation. In order to understand the dynamic mechanisms that mediate craniosynostosis, we needed to investigate the biologic processes, before, during and after suture fusion. Since clinical specimens limit our investigation to the time at which the samples are excised, we have employed murine models to examine the cascading events that lead to cranial suture fusion. These models have enabled us to dissect, isolate and understand the individual roles of the dura mater, pericranium, suture mesenchyme and osteogenic fronts.

Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms.

Transforming growth factor-betas (TGF-beta) have been demontstrated to be upregulated during osteoblast function in vitro and during cranial suture fusion in vivo. The authors hypothesized that spontaneous reossification of calvarial defects was also associated with upregulation of TGF-beta. The present study was designed to (1) evaluate the concept of a critical-size defect within the calvaria in an adult guinea pig model and (2) investigate the association between the ossification of calvarial defects and TGF-beta upregulation. Paired circular parietal defects with diameters of 3 and 5 mm and single parietal defects with diameters of 8 or 12 mm were made in 45 six-month-old skeletally mature guinea pigs. Three animals per defect size were killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method using an image analysis system in which the thickness of new bone was taken into account. Immunohistochemistry was performed using rabbit polyclonal antibodies to localize TGF-beta1, -beta2, and -beta3. All specimens were photographed, and the intensity of immunostaining was graded based on subjective photographic assessment by three independent reviewers. No defect demonstrated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of new bone, indicating these defects to be less than critical size. However, new bone formation in the 5-mm defects never exceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reaching a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks, and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocalization demonstrated osteogenic fronts at the advancing bone edge and the endocranial side, in which the osteoblasts stained strongly for all isoforms of TGF-beta. The intensity of osteoblast expression waned considerably after the majority of the defect had reossified. These data indicate that histometric analysis based on cross-sectional area more accurately reflects the osteogenic potential of a cranial defect than does linear inspection of defect closure. Although the interpretation of immunolocalization studies is highly subjective, independent assessment by three reviewers indicates that isoforms of TGF-beta were upregulated during a limited "window" of time corresponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts.

Integration of FGF and TWIST in calvarial bone and suture development.

Mutations in the FGFR1-FGFR3 and TWIST genes are known to cause craniosynostosis, the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. We have previously shown that Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones and that, when FGF is applied via beads to the osteogenic fronts, suture closure is accelerated (Kim, H.-J., Rice, D. P. C., Kettunen, P. J. and Thesleff, I. (1998) Development 125, 1241-1251). In order to investigate further the role of FGF signalling during mouse calvarial bone and suture development, we have performed detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 being expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 showed a more restricted expression pattern being primarily expressed in the sutural mesenchyme between the osteogenic fronts. We also carried out a detailed expression analysis of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 were expressed by early preosteoblasts, in patterns that overlapped those of the FGF ligands, but as these cells differentiated their expression dramatically decreased. Signalling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induced Twist and inhibited Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulated Id1. Id1 is a dominant negative HLH thought to inhibit basic HLH such as Twist. In Drosophila, the FGF receptor FR1 is known to be downstream of Twist. We demonstrated that in Twist(+/)(-) mice, FGFR2 protein expression was altered. We propose a model of osteoblast differentiation integrating Twist and FGF in the same pathway, in which FGF acts both at early and late stages. Disruption of this pathway may lead to craniosynostosis.

Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.

The composite structure of the mammalian skull, which forms predominantly via intramembranous ossification, requires precise pre- and post-natal growth regulation of individual calvarial elements. Disturbances of this process frequently cause severe clinical manifestations in humans. Enhanced DNA binding by a mutant MSX2 homeodomain results in a gain of function and produces craniosynostosis in humans. Here we show that Msx2-deficient mice have defects of skull ossification and persistent calvarial foramen. This phenotype results from defective proliferation of osteoprogenitors at the osteogenic front during calvarial morphogenesis, and closely resembles that associated with human MSX2 haploinsufficiency in parietal foramina (PFM). Msx2-/- mice also have defects in endochondral bone formation. In the axial and appendicular skeleton, post-natal deficits in Pth/Pthrp receptor (Pthr) signalling and in expression of marker genes for bone differentiation indicate that Msx2 is required for both chondrogenesis and osteogenesis. Consistent with phenotypes associated with PFM, Msx2-mutant mice also display defective tooth, hair follicle and mammary gland development, and seizures, the latter accompanied by abnormal development of the cerebellum. Most Msx2-mutant phenotypes, including calvarial defects, are enhanced by genetic combination with Msx1 loss of function, indicating that Msx gene dosage can modify expression of the PFM phenotype. Our results provide a developmental basis for PFM and demonstrate that Msx2 is essential at multiple sites during organogenesis.

Apoptosis in murine calvarial bone and suture development.

To study the possible role of apoptosis in calvarial bone and suture development, terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) was performed on whole mount and sectioned calvariae from mice aged between E14 and P6. We also analyzed by in situ hybridization the expression of Msx2, Bmp4 and Bmp7 genes, which are known to act in conserved signaling pathways leading to apoptosis. We found TUNEL-positive cells from E16 onwards in the calvarial bones, intervening sutures and fontanelles. TUNEL-positive osteoblasts and preosteoblasts were identified at or close to the osteogenic fronts, areas of intense osteogenic activity, with TUNEL-positive mesenchymal cells located in the midsutural mesenchyme. TUNEL-positive osteoclasts and osteocytes were also observed in a sporadic fashion, as well as TUNEL-positive dural cells. Msx2 was expressed in the sutural mesenchyme and the dura mater. Bmp4 was expressed in the developing bone, underlying dura mater, the osteogenic fronts, and also weakly in the sutural mesenchyme. Bmp7 was detected at the same locations as Bmp4 but with noticeably stronger intensity in the meninges and overlying epidermis. We propose that this apoptosis is part of normal suture development, and is integral to the balance between bone formation and resorption, so that abnormal apoptosis may lead to premature (Craniosynostosis) or delayed (Cleidocranial dysplasia) suture closure.

EctopicMsx2Overexpression Inhibits andMsx2Antisense Stimulates Calvarial Osteoblast Differentiation

Msx2is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression ofMsx2mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels ofMsx2protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression ofMsx2prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisenseMsx2RNA decreased proliferation and accelerated differentiation. In other studies, we showed that theMsx2promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in whichMsx2prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.

Coronal suture response to distraction osteogenesis in rabbits with delayed-onset craniosynostosis.

Recent studies have identified a subpopulation of persons with craniosynostosis who exhibit progressive or delayed-onset synostosis and mild cranial vault deformities. These persons may be good candidates for nonextirpation distraction osteogenesis. The present studies were designed to determine force-displacement parameters and assess the effects of distraction osteogenesis on coronal suture growth and morphologic characteristics in a rabbit model with congenital, delayed-onset craniosynostosis. Data were collected from a total of 178 rabbits: 71 normal controls; 16 normal controls with distraction; 72 with delayed-onset coronal suture synostosis; and 19 with delayed-onset coronal suture synostosis and distraction. At 10 days of age, all rabbits had amalgam markers placed on both sides of the coronal suture. In the force-displacement study, force-displacement distractors were placed across the coronal suture and distracted acutely for 1.0 mm at 42 days of age. Force-displacement curves for the coronal suture were best described by a third-order polynomial regression equation for both normal and synostosed groups. Significant differences (P < 0.05) were found in the mean force necessary to distract a normal suture 1 mm in distance (13.72 kg) compared with a suture with delayed-onset synostosis (48.39 kg). A significant (P < 0.05) relationship was also found between the extent of synostosis and the distractive force in rabbits with delayed-onset synostosis. In the distraction study, internal distractors were fixed across the coronal suture at 25 days of age and percutaneously and intermittently activated at an average of 0.11 mm/day for 42 days (4.54 mm total). Serial radiographs were taken at 10, 25, 42, and 84 days of age. Results revealed that rabbits with delayed-onset synostosis and distraction had significantly (P < 0.01) more coronal suture growth rates compared with rabbits with delayed-onset synostosis and no distraction. Coronal sutures were harvested at 84 days of age for qualitative histologic examination. Normal, distracted coronal sutures showed widened sutural ligaments and thin, active osteogenic fronts. In contrast, distracted coronal sutures from rabbits with delayed-onset synostosis showed narrowed sutural ligaments, thickened and blunt osteogenic fronts, and increased collagen and bony matrix deposition compared with controls. Results suggest that distraction osteogenesis without corticotomy may be a treatment alternative in persons with progressive, delayed-onset synostosis. However, these preliminary data also suggest that distractive forces may accelerate or stimulate osteogenesis differentially in persons with craniosynostosis, possibly through an underlying genetic disorder of bone and cytokine regulation. These differential osteogenic responses to distraction, if validated clinically, will need to be taken into account when planning distraction rate and rhythm protocols for patients with craniosynostosis.

Human NELL-1 expressed in unilateral coronal synostosis.

Surgical correction of unilateral coronal synostosis offers a unique opportunity to examine the molecular differences between an abnormal and a normal cranial suture. We isolated and identified a cDNA fragment whose expression was up-regulated in the premature fusing and fused coronal sutures, as compared with normal coronal sutures. The nucleotide sequence of the full-length cDNA of this gene, human NELL-1, has approximately 61% homology with the chicken Nel gene. Both chicken Nel and human NELL-1 are comprised of six epidermal growth factor-like repeats. The human NELL-1 messages were localized primarily in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins, and within the condensing mesenchymal cells of newly formed bone in sites of premature sutural fusion. Human multiorgan tissue mRNA blot showed that NELL-1 was specifically expressed in fetal brain but not in fetal kidney, liver, or lung. We also showed that Nell-1 was expressed in rat calvarial osteoprogenitor cells and was largely absent in rat tibiae and fibroblast cell cultures. In conclusion, our data suggest that the NELL-1 gene is preferentially expressed in cranial intramembranous bone and neural tissue (both of neural crest cell origin) and is up-regulated during unilateral premature closure of the coronal suture. The precise role of this gene is unknown.

Msx2 gene dosage influences the number of proliferative osteogenic cells in growth centers of the developing murine skull: a possible mechanism for MSX2-mediated craniosynostosis in humans.

Throughout its complex morphogenesis, the vertebrate skull must at once protect the brain and expand to accommodate its growth. A key structural adaptation that allows this dual role is the separation of the bony plates of the skull with sutures, fibrous joints that serve as growth centers and allow the calvarial bones to expand as the brain enlarges. Craniosynostosis, the premature fusion of one or more calvarial bones with consequent abnormalities in skull shape, is a common developmental anomaly that disrupts this process. We found previously that a single amino acid substitution in the homeodomain of the human MSX2 gene is associated with the autosomal dominant disorder craniosynostosis, Boston type. This mutation enhances the affinity of Msx2 for its target sequence, suggesting that the mutation acts by a dominant positive mechanism. Consistent with this prediction, we showed that general overexpression of Msx2 under the control of the broadly expressed CMV promoter causes the calvarial bones to invade the sagittal suture. Here we use tissue-specific overexpression of Msx2 within the calvarial sutures to address the developmental mechanisms of craniosynostosis and skull morphogenesis. We demonstrate that a segment of the Msx2 promoter directs reporter gene expression to subsets of cells within the sutures. In late embryonic and neonatal stages, this promoter is expressed in undifferentiated mesenchymal cells medial to the growing bone. By P4, promoter activity is reduced in the suture, exhibiting a punctate pattern in undifferentiated osteoblastic cells in the outer margin of the osteogenic front. Overexpression of Msx2 under the control of this promoter is sufficient to enhance parietal bone growth into the sagittal suture by P6. This phenotype is preceded by an increase in both the number and the BrdU labeling of osteoblastic cells in the osteogenic fronts of the calvarial bones. These findings suggest that an important early event in MSX2-mediated craniosynostosis in humans is a transient retardation of osteogenic cell differentiation in the suture and a consequent increase in the pool of osteogenic cells.

A rabbit model of human familial, nonsyndromic unicoronal suture synostosis. I. Synostotic onset, pathology, and sutural growth patterns.

Poswillo has stated, "The more severe anomalies of the calvaria, such as plagiocephaly, Crouzon [syndrome], and Apert syndrome still defy explanation, in the absence of an appropriate animal system to study" (p. 207). This two-part study reviews data from a recently developed colony of New Zealand white rabbits with familial, nonsyndromic unilateral coronal suture synostosis. Part 1 presents pathological findings and compensatory sutural growth data from 109 normal rabbits and 82 craniosynostotic rabbits from this colony. Synostotic foci, onset, and progression were described in the calvariae from 102 staged (fetal days 21, 25, 27, 33; term = 30 days) fetuses (39 normal, 63 synostosed). Calvarial suture growth patterns from 10 to 126 days of age were assessed from serial radiographs obtained from 89 rabbits (70 normal rabbits and 19 rabbits with unicoronal suture synostosis) with amalgam bone marker implants. Perinatal results revealed that by fetal day 25 the synostotic focal point in synostotic rabbits consistently originated from the endocortical surface of the calvaria in the middle of the coronal suture at a presumed high-tension, interdigitating zone. Histological analysis revealed hyperostotic osteogenic fronts on the affected side compared with the unaffected side. Postnatal sutural growth data revealed a predictable pattern of plagiocephaly (contralateral coronal sutures growing more than ipsilateral sutures and ipsilateral frontonasal and anterior lambdoidal sutures growing more than contralateral sutures), which resulted in early cranial vault deformities and a double "S" shape torquing towards the affected side. The advantages and disadvantages of these rabbits as a model for human familial, nonsyndromic unicoronal suture synostosis are discussed, especially in light of recent cytokine and genetic findings from human craniosynostotic studies.

FGF-, BMP- and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis and calvarial bone development.

The development of calvarial bones is tightly co-ordinated with the growth of the brain and needs harmonious interactions between different tissues within the calvarial sutures. Premature fusion of cranial sutures, known as craniosynostosis, presumably involves disturbance of these interactions. Mutations in the homeobox gene Msx2 as well as the FGF receptors cause human craniosynostosis syndromes. Our histological analysis of mouse calvarial development demonstrated morphological differences in the sagittal suture between embryonic and postnatal stages. In vitro culture of mouse calvaria showed that embryonic, but not postnatal, dura mater regulated suture patency. We next analysed by in situ hybridisation the expression of several genes, which are known to act in conserved signalling pathways, in the sagittal suture during embryonic (E15-E18) and postnatal stages (P1-P6). Msx1 and Msx2 were expressed in the sutural mesenchyme and the dura mater. FGFR2(BEK), as well as Bmp2 and Bmp4, were intensely expressed in the osteogenic fronts and Bmp4 also in the mesenchyme of the sagittal suture and in the dura mater. Fgf9 was expressed throughout the calvarial mesenchyme, the dura mater, the developing bones and the overlying skin, but Fgf4 was not detected in these tissues. Interestingly, Shh and Ptc started to be expressed in patched pattern along the osteogenic fronts at the end of embryonic development and, at this time, the expression of Bmp4 and sequentially those of Msx2 and Bmp2 were reduced, and they also acquired patched expression patterns. The expression of Msx2 in the dura mater disappeared after birth. <P> FGF and BMP signalling pathways were further examined in vitro, in E15 mouse calvarial explants. Interestingly, beads soaked in FGF4 accelerated sutural closure when placed on the osteogenic fronts, but had no such effect when placed on the mid-sutural mesenchyme. BMP4 beads caused an increase in tissue volume both when placed on the osteogenic fronts and on the mid-sutural area, but did not effect suture closure. BMP4 induced the expression of both Msx1 and Msx2 genes in sutural tissue, while FGF4 induced only Msx1. We suggest that the local application of FGF on the osteogenic fronts accelerating suture closure in vitro, mimics the pathogenesis of human craniosynostosis syndromes in which mutations in the FGF receptor genes apparently cause constitutive activation of the receptors. Taken together, our data suggest that conserved signalling pathways regulate tissue interactions during suture morphogenesis and intramembranous bone formation of the calvaria and that morphogenesis of mouse sagittal suture is controlled by different molecular mechanisms during the embryonic and postnatal stages. Signals from the dura mater may regulate the maintenance of sutural patency prenatally, whereas signals in the osteogenic fronts dominate after birth.

Molecular signaling in suture development

The aim of this study (1) was to develop an in vitro organ culture system to facilitate the study of molecular signaling in the developing calvarial bones and sutures. Calvaria of mice aged 15 embryonic days (E15) were dissected free under a stereomicroscope using ophthalmic scissors. Explants were placed in a Trowell-type organ culture and observed for 1-4 days. The explants developed in a manner consistent with suture development in vivo. The opposing parietal bone osteogenic fronts (OFs) approximated but did not fuse. In vitro osteoblast activity, analyzed by in situ hybridization for bone sialoprotein, correlated well with in vivo development. In situ hybridization on both whole mount and sectioned tissue was carried out to detect mRNA for fibroblast growth factor receptors (Fgfr's), Fgf's and Msx's, genes known to cause human craniosynostosis or thought to be involved in calvarial development. To test whether growth factors interacted with each other, beads impregnated with the molecule under investigation were placed in various locations and their effects analyzed. Bek, a splicing alternative of Fgfr2, was intensely expressed in the OFs of parietal bones of E15 and E17 mouse embryos. Postnatally, its intensity diminished. Fgf2, a ligand known to bind to Fgfr2, was intensely expressed in the sutural mesenchyme between the OFs at 15 embryonic days; this had declined at 17 embryonic days, and transcripts were not detected postnatally. Msx1 was expressed in the sutural mesenchyme and the underlying dura mater during embryonic and postnatal stages. Embryonically, Msx2 was intensely expressed in locations similar to those of Msx1. However, after birth the expression of Msx2 was dramatically diminished in the mesenchyme, and it completely disappeared from the dura mater. Beads soaked in FGF4 accelerated sutural closure when placed on the osteogenic fronts but had no such effect when placed on the mid-sutural mesenchyme. BMP4 beads caused an increase in tissue volume but did not effect suture closure. BMP4 induced the expression of both Msx1 and Msx2 genes in sutural tissue, while FGF4 induced only Msx1. In conclusion, we have developed microdissection techniques and an organ culture system for the developing mouse calvaria. We suggest that the local application of FGF on the osteogenic fronts, which accelerates suture closure, mimics the pathogenesis of human craniosynostosis syndromes in which mutations in the FGF receptor genes apparently cause constitutive activation of the receptors.

Coronal Suture Pathology and Synostotic Progression in Rabbits with Congenital Craniosynostosis

The purpose of the present study was to describe coronal suture pathology and cross sectional synostotic progression in an inbred strain of rabbits with congenital craniosynostosis. Calvaria from 102 perinatal rabbits (39 unaffected; 63 bilateral or unilateral synostosis) were collected at fetal days 21 (n = 12), 25 (n = 20), 27 (n = 22), 30 (term) (n = 32), and 3 days post-term (n = 16) for gross morphologic and histologic examination. Synostotic foci, the extent of relative bony bridging, and suture morphology were evaluated qualitatively and quantitatively. Of the 204 coronal sutures examined, 91 sutures were synostosed, and 113 were patent. All synostosed sutures showed similar foci by day 25, which originated as bony bridges in the middle of each suture on the ectocortic surface. Bony bridging width increased significantly (p < .001) from day 25 through 3 days post-term, and was best described by a linear regression equation. Osteogenic front areas of synostosed sutures were up to 2.5 times greater than patent sutures in term fetuses. Findings demonstrate that coronal suture synostosis in the congenital rabbit model (1) begins early during suture morphogenesis (before 25 days of gestation); (2) consistently radiates from a single focus corresponding to a normal interdigitating region (i.e., a high-tension environment); (3) varies in onset and rate as evidenced by low R2 value between age and extent of bony bridging; and (4) is the result of early hyperostosis of the osteogenic fronts and sutural agenesis. A number of possible pathogenetic mechanisms are discussed.

Coronal suture pathology and synostotic progression in rabbits with congenital craniosynostosis.

The purpose of the present study was to describe coronal suture pathology and cross sectional synostotic progression in an inbred strain of rabbits with congenital craniosynostosis. Calvaria from 102 perinatal rabbits (39 unaffected; 63 bilateral or unilateral synostosis) were collected at fetal days 21 (n = 12), 25 (n = 20), 27 (n = 22), 30 (term) (n = 32), and 3 days post-term (n = 16) for gross morphologic and histologic examination. Synostotic foci, the extent of relative bony bridging, and suture morphology were evaluated qualitatively and quantitatively. Of the 204 coronal sutures examined, 91 sutures were synostosed, and 113 were patent. All synostosed sutures showed similar foci by day 25, which originated as bony bridges in the middle of each suture on the ectocortic surface. Bony bridging width increased significantly (p < .001) from day 25 through 3 days post-term, and was best described by a linear regression equation. Osteogenic front areas of synostosed sutures were up to 2.5 times greater than patent sutures in term fetuses. Findings demonstrate that coronal suture synostosis in the congenital rabbit model (1) begins early during suture morphogenesis (before 25 days of gestation); (2) consistently radiates from a single focus corresponding to a normal interdigitating region (i.e., a high-tension environment); (3) varies in onset and rate as evidenced by low R2 value between age and extent of bony bridging; and (4) is the result of early hyperostosis of the osteogenic fronts and sutural agenesis. A number of possible pathogenetic mechanisms are discussed.

Light and electron microscopy of the new born sagittal suture.

Morphogenesis and maturation of the sagittal suture in newborn C57B1/6J strain mice were studied using light and electron microscopy. Morphodifferentiation of the murine parietal bones progresses radially with the interposed sagittal suture, assuming a greater level of maturity at birth at a midpoint along its length. The presumptive suture develops in a sulcus, deeper posteriorly, more shallow anteriorly. Cells at the osteogenic front (OF) are distinguished from the surrounding fibrocytic cells by a number of distinctive characteristics: 1. increased cytoplasmic density; 2. extensive endoplasmic reticulum; 3. dispersed nuclear chromatin aggregates; 4. extensive surface projections; 5. close approximation. Mineralization of the developing parietal bone occurs extracellularly with the initial deposits of apatite crystals exhibiting no oriented relationship to either cellular or extracellular fibrillar elements. The majority of collagen fibers lie superior and inferior to the presumptive suture, oriented anteroposteriorly with their long axes parallel to the ectocranial surface. Other fiber bundles more intimately associated with the developing suture display a more random orientation.