Cranial Suture Biology of the Aleutian Island Inhabitants

Research on cranial suture biology suggests there is biological and taxonomic information to be garnered from the heritable pattern of suture synostosis. Suture synostosis along with brain growth patterns, diet, and biomechanical forces influence phenotypic variability in cranial vault morphology. This study was designed to determine the pattern of ectocranial suture synostosis in skeletal populations from the Aleutian Islands. We address the hypothesis that ectocranial suture synostosis pattern will differ according to cranial vault shape. Ales Hrdlicka identified two phenotypes in remains excavated from the Aleutian Island. The Paleo‐Aleutians, exhibiting a dolichocranic phenotype with little prognathism linked to artifacts distinguished from later inhabitants, Aleutians, who exhibited a brachycranic phenotype with a greater amount of prognathism. A total of 212 crania representing Paleo‐Aleuts and Aleutian as defined by Hrdlicka were investigated for suture synostosis pattern following standard methodologies. Comparisons were performed using Guttmann analyses. Results revealed similar suture fusion patterns for the Paleo‐Aleut and Aleutian, a strong anterior to posterior pattern of suture fusion for the lateral‐anterior suture sites, and a pattern of early termination at the sagittal suture sites for the vault. These patterns were found to differ from that reported in the literature. Because these two populations with distinct cranial shapes exhibit similar patterns of suture synostosis it appears pattern is independent of cranial shape in these populations of Homo sapiens. These findings suggest that suture fusion patterns may be population dependent and that a standardized methodology, using suture fusion to determine age‐at‐death, may not be applicable to all populations. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Cranial sutures and bones: growth and fusion in relation to masticatory strain

Cranial bones and sutures are mechanically loaded during mastication. Their response to masticatory strain, however, is largely unknown, especially in the context of age change. Using strain gages, this study investigated masticatory strain in the posterior interfrontal and the anterior interparietal sutures and their adjacent bones in 3- and 7-month-old miniature swine (Sus scrofa). Double-fluorochrome labeling of these animals and an additional 5-month group was used to reveal suture and bone growth as well as features of suture morphology and fusion. With increasing age, the posterior interfrontal suture strain decreased in magnitude and changed in pattern from pure compression to both compression and tension, whereas the interparietal suture remained in tension and the magnitude increased unless the suture was fused. Morphologically, the posterior interfrontal suture was highly interdigitated at 3 months and then lost interdigitation ectocranially in older pigs, whereas the anterior interparietal suture remained butt-ended. Mineralization apposition rate (MAR) decreased with age in both sutures and was unrelated to strain. Bone mineralization was most vigorous on the ectocranial surface of the frontal and the parietal bones. Unlike the sutures, with age bone strain remained constant while bone MARs significantly increased and were correlated with bone thickness. Fusion had occurred in the interparietal suture of some pigs. In all cases fusion was ectocranial rather than endocranial. Fusion appeared to be associated with increased suture strain and enhanced bone growth on the ectocranial surface. Collectively, these results indicate that age is an important factor for strain and growth of the cranium. .     

Erk pathway and activator protein 1 play crucial roles in FGF2-stimulated premature cranial suture closure.

Cranial sutures are an important growth center of the cranial bones, and the suture space must be maintained to permit the cranial adjustments needed to accommodate brain growth. Craniosynostosis, characterized by premature suture closure, mainly results from mutations that generate constitutively active fibroblast growth factor (FGF) receptors. FGF signaling, thus, is responsible for the pathogenesis of craniosynostosis. Even though FGF activates many different signaling pathways, the one involved in premature suture closure has not been defined. We observed that placing FGF2-soaked bead on the osteogenic fronts of cultured mouse calvaria accelerates cranial suture closure and strongly induces the expression of osteopontin, an early marker of differentiated osteoblasts. FGF2 treatment also induced fos and jun mRNAs and later increased the nuclear levels of activator protein 1 (AP1). FGF2 stimulates the expression of osteopontin by inducing expression of AP1, which then binds to its response element in the osteopontin promoter. Blocking of the Erk pathway by PD98059 suppressed the AP1 and osteopontin expression stimulated by FGF2. Coincidently, blocking of the Erk pathway also significantly retarded FGF2-accelerated cranial suture closure. Thus, the Erk pathway mediates FGF/FGF receptor-stimulated cranial suture closure, probably by stimulating synthesis of AP1 that then stimulates the differentiation of osteoblasts.     

Regulation of cranial suture morphogenesis

The cranial sutures are the primary sites of bone formation during skull growth. Morphogenesis and phenotypic maintenance of the cranial sutures are regulated by tissue interactions, especially those with the underlying dura mater. Removal of the dura mater in fetuses causes abnormal suture development and premature suture obliteration. The dura mater interacts with overlying tissues of the cranial vault by providing: (1) intercellular signals, (2) mechanical signals and (3) cells, which undergo transformation and migrate to the suture. The intercellular signaling governing suture development employs the fibroblast growth factors (FGFs). In rats during formation of the sutures in the fetus, FGF-1 is localized mainly in the dura mater, while other FGFs are expressed in the overlying tissues. By birth, FGF-2 largely replaces FGF-1 in the dura mater. FGFs present in the calvaria bind either the IIIb or IIIc mRNA splice variants of the FGF receptors (FGFRs) 1, 2, or 3. Monoclonal antibodies to the b variant of FGFR2 were used to determine the distribution of FGFR2IIIb during suture development and its extracellular localization. FGFR2IIIb is present in association with mature osteoblasts and osteogenic precursor cells of the suture in the fetus. Ectodomains of FGFR2IIIb, the products of proteolytic cleavage of the receptors, were present throughout the extracellular matrix of sutures resisting obliteration (coronal and sagittal), but absent from the core of sutures undergoing normal fusion (posterior intrafrontal). This observation is consistent with a possible mechanism, in which truncated receptors bind FGFs, thus regulating free FGF available to nearby cells. Mechanical signaling in the calvaria results from tensional forces in the dura mater generated during rapid expansion of the neurocranium. Posterior intrafrontal sutures of rats, which fuse between days 16 and 24, were subjected to cyclical tensional forces in vitro. Significant delay in the timing of suture fusion and increases in the expression domains of FGFR1 and 2 were observed, demonstrating the sensitivity of suture patency to mechanical signals and a possible role of the FGF system in mediating such stimuli. Finally, cells of the dura mater beneath the intrafrontal and sagittal sutures were observed to undergo a morphological transformation to a dendritic morphology and migrate into the suture mesenchyme between days 10 and 16 of development. This process may participate in suture and bone morphogenesis and influence the patency of the sutures along the anterior-posterior axis.     

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.     

Changes in Biomechanical Strain and Morphology of Rat Calvarial Sutures and Bone After Tgf‐β3 Inhibition of Posterior Interfrontal Suture Fusion

Craniofacial sutures are bone growth fronts that respond and adapt to biomechanical environments. Little is known of the role sutures play in regulating the skull biomechanical environment during patency and fusion conditions, especially how delayed or premature suture fusion will impact skull biomechanics. Tgf‐β3 has been shown to prevent or delay suture fusion over the short term in rat skulls, yet the long‐term patency or its consequences in treated sutures is not known. It was therefore hypothesized that Tgf‐β3 had a long‐term impact to prevent suture fusion and thus alter the skull biomechanics. In this study, collagen gels containing 3 ng Tgf‐β3 were surgically placed superficial to the posterior interfrontal suture (IFS) and deep to the periosteum in postnatal day 9 (P9) rats. At P9, P24, and P70, biting forces and strains over left parietal bone, posterior IFS, and sagittal suture were measured with masticatory muscles bilaterally stimulated, after which the rats were sacrificed and suture patency analyzed histologically. Results demonstrated that Tgf‐β3 treated sutures showed less fusion over time than control groups, and strain patterns in the skulls of the Tgf‐β3‐treated group were different from that of the control group. Although bite force increased with age, no alterations in bite force were attributable to Tgf‐β3 treatment. These findings suggest that the continued presence of patent sutures can affect strain patterns, perhaps when higher bite forces are present as in adult animals. Anat Rec,, 2012. © 2012 Wiley Periodicals, Inc.     

Beyond the closed suture in apert syndrome mouse models: Evidence of primary effects of FGFR2 signaling on facial shape at birth

Apert syndrome is a congenital disorder caused mainly by two neighboring mutations on fibroblast growth factor receptor 2 (FGFR2). Premature closure of the coronal suture is commonly considered the identifying and primary defect triggering or preceding the additional cranial malformations of Apert phenotype. Here we use two transgenic mouse models of Apert syndrome, Fgfr2^+/S252W and Fgfr2^+/P253R, to explore variation in cranial phenotypes in newborn (P0) mice. Results show that the facial skeleton is the most affected region of the cranium. Coronal suture patency shows marked variation that is not strongly correlated with skull dysmorphology. The craniofacial effects of the FGFR2 mutations are similar, but Fgfr2^+/S252W mutant mice display significantly more severe dysmorphology localized to the posterior palate. Our results demonstrate that coronal suture closure is neither the primary nor the sole locus of skull dysmorphology in these mouse models for Apert syndrome, but that the face is also primarily affected. Developmental Dynamics 239:3058–3071, 2010. © 2010 Wiley‐Liss, Inc.     

Rescue of coronal suture fusion using transforming growth factor-beta 3 (Tgf-beta 3) in rabbits with delayed-onset craniosynostosis

Craniosynostosis results in cranial deformities and increased intracranial pressure, which pose extensive and recurrent surgical management problems. Developmental studies in rodents have shown that low levels of transforming growth factor-beta 3 (Tgf-beta 3) are associated with normal fusion of the interfrontal (IF) suture, and that Tgf-beta 3 prevents IF suture fusion in a dose-dependent fashion. The present study was designed to test the hypothesis that Tgf-beta 3 can also prevent or "rescue" fusing sutures in a rabbit model with familial craniosynostosis. One hundred coronal sutures from 50 rabbits with delayed-onset, coronal suture synostosis were examined in the present study. The rabbits were divided into five groups of 10 rabbits each: 1) sham controls, 2) bovine serum albumin (BSA, 500 ng) low-dose protein controls, 3) low-dose Tgf-beta 3 (500 ng), 4) high-dose BSA (1,000 ng) controls, and 5) high-dose Tgf-beta 3 (1,000 ng). At 10 days of age, radiopaque amalgam markers were implanted in all of the rabbits on either side of the coronal suture to monitor sutural growth. At 25 days of age, the BSA or Tgf-beta 3 was combined with a slow-absorbing collagen vehicle and injected subperiosteally above the coronal suture. Radiographic results revealed that high-dose Tgf-beta 3 rabbits had significantly greater (P < 0.05) coronal suture marker separation than the other groups. Histomorphometric analysis revealed that high-dose Tgf-beta 3 rabbits also had patent coronal sutures and significantly (P < 0.01) greater sutural widths and areas than the other groups. The results suggest that there is a dose-dependent effect of TGF-beta 3 on suture morphology and area in these rabbits, and that the manipulation of such growth factors may have clinical applications in the treatment of craniosynostosis.     

Suture formation,premature sutural fusion,and suture default zones in Apert syndrome

On the basis of our studies, we postulate that suture formation in Apert syndrome is related to the relative maturity of abutting calvarial bones. The fused coronal suture, a consistent manifestation at birth, develops first because the ossification centers of the frontal and parietal bones are in intimate contact early during intrauterine life. Calvarial immaturity and the megalencephalic brain characteristic of the Apert syndrome appear to work in concert to produce a widely patent midline calvarial defect extending from the glabella to the posterior fontanelle. Because sagittal growth in the coronal sutures cannot take place, the megalencephalic brain grows upward and laterally, and bulges forward through the midline defect. The defect fills in by coalescence of bony islands without proper suture formation because the gap to be bridged is so great that the time window for developing sutural interdigitations may have closed. Other sutures, such as the lambdoid, squamosal, and sphenotemporal, develop with normal interdigitations because abutting bone margins are in close enough proximity to permit suture formation. © 1996 Wiley-Liss, Inc.     

Timing of ectocranial suture activity in Gorilla gorilla as related to cranial volume and dental eruption

Research has shown that Pan and Homo have similar ectocranial suture synostosis patterns and a similar suture ontogeny (relative timing of suture fusion during the species ontogeny). This ontogeny includes patency during and after neurocranial expansion with a delayed bony response associated with adaptation to biomechanical forces generated by mastication. Here we investigate these relationships for Gorilla by examining the association among ectocranial suture morphology, cranial volume (as a proxy for neurocranial expansion) and dental development (as a proxy for the length of time that it has been masticating hard foods and exerting such strains on the cranial vault) in a large sample of Gorilla gorilla skulls. Two-hundred and fifty-five Gorilla gorilla skulls were examined for ectocranial suture closure status, cranial volume and dental eruption. Regression models were calculated for cranial volumes by suture activity, and Kendall's tau (a non-parametric measure of association) was calculated for dental eruption status by suture activity. Results suggest that, as reported for Pan and Homo, neurocranial expansion precedes suture synostosis activity. Here, Gorilla was shown to have a strong relationship between dental development and suture activity (synostosis). These data are suggestive of suture fusion extending further into ontogeny than brain expansion, similar to Homo and Pan. This finding allows for the possibility that masticatory forces influence ectocranial suture morphology.     

Raman imaging demonstrates FGF2-induced craniosynostosis in mouse calvaria

Craniosynostosis is a severe craniofacial disease where one or more sutures, the fibrous tissue that lies between the cranial bones, fuses prematurely. Some craniosynostosis syndromes are known to be caused by mutations in fibroblast growth factor (FGF) receptors. Mutated FGF receptors are thought to cause constitutive signaling. In this study, heparin acrylic beads released fibroblast growth factor 2 (FGF2) to mimic constitutive signaling by mutated receptors, delivering FGF2 in addition to already existing normal tissue amounts. Fetal day 18.5 mouse sutures were treated with FGF2-soaked beads and cultured in serum free media for 48 h. We have shown previously that this treatment leads to fusion and increased Msx2 expression, but here we use near-infrared Raman imaging to simultaneously examine the mineral components and matrix components of cranial tissue while providing light microscopic spatial information. FGF2-treated mouse sutures show increased v1 phosphate and v1 carbonate bandwidths, indicating a slightly chemically modified mineral being rapidly deposited. In addition, FGF2-treated mouse sutures show a marked increase in mineral-to-matrix ratios compared to control mouse sutures, typical of increased mineralization.     

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 stereo-photogrammetrical analysis. The parietal bones were found to rotate laterally upward (3°), while the frontal bones rotated downward (2°) relative to their contralaterals. The frontal bones rotated rostrally upward (12°) and outward (3°) as well as laterally downward (5°) 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° 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.     

Bone morphogenetic protein is required for fibroblast growth factor 2-dependent later-stage osteoblastic differentiation in cranial suture cells

Background: Understanding the pathophysiological process of calvarial bones development is important for the treatments on relative diseases such as craniosynostosis. While, the role of fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) and how they interacted in osteoblast differentiation remain unclear. Methods: we digested bone fragments around the coronal and sagittal sutures from newborn rats to harvest suture cells. Markers expression at different osteoblast differentiation stage was analyzed by increasing FGF2 concentration and BMP2 blocking in these cells. Results: BMP2 expression could be stimulated by FGF2 in a dose and time dependent manner. FGF2 stimulation may decrease early marker of osteoblast differentiation (collagen type-1, COL-1) and increase the expression of continuously-expressed or late markers (alkaline phosphatase, ALP; osteocalcin, OC and bone sialoprotein, BSP) to accelerate mineralization. Inhibition of BMP2 signaling by Noggin weakens the effect of FGF2 on induction of later-stage osteoblastic differentiation of cranial suture cells. Conclusion: Our data suggest that BMP2 signaling is required for FGF2-dependent induction of later-stage of cranial suture cell osteoblastic differentiation.     

Craniosynostosis: genes and mechanisms

Enlargement of the skull vault occurs by appositional growth at the fibrous joints between the bones, termed cranial sutures. Relatively little is known about the developmental biology of this process, but genetically determined disorders of premature cranial suture fusion (craniosynostosis) provide one route to the identification of some of the key molecules involved. Mutations of the MSX2, FGFR1, FGFR2, FGFR3 and TWIST genes yield new insights, both into normal and abnormal cranial suture biogenesis and into problems of broad interest, such as the conservation of molecular pathways in development, and mechanisms of mutation and dominance.      

Cell proliferation and osteogenic differentiation of growing pig cranial sutures

Bone growth at the cranial sutures relies on proliferation of osteogenic progenitor cells and/or differentiation of osteoblasts. The current study was undertaken to assess these events in relation to suture growth and fusion. A total of 21 pigs, divided into three age groups (0.5-1.5 months, 3-4 months and 5-7 months), were used for immunohistochemical evaluation of cell proliferation (BrdU) and osteogenic differentiation (Cbfa1/Runx2) in the interfrontal and interparietal sutures. Proliferation and osteogenic differentiation were both more prominent near the bone fronts than in the central zone. With age, both proliferation and osteogenic differentiation diminished. Proliferation ceased on the endocranial (dura mater) side by the age of 3-4 months. Proliferation on the pericranial side was accompanied by active bone formation and initiation of suture fusion from this side. In conclusion, (1) decreased suture bone growth with age reflects decreased cell proliferation and probably also osteogenic differentiation, and (2) suture fusion occurs from the pericranial side where activity remains relatively high.