S Warren, M Longaker
cranial suture biology, craniofacial disorders, development, dura mater, medicine, osteoblast, plastic surgery
S Warren, M Longaker. Advances In Murine Cranial Suture Research. The Internet Journal of Neurosurgery. 2000 Volume 1 Number 2.
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.
Although murine and human craniofacial characteristics are obviously different, there appears to be tremendous conservation in the assembly of embryonic cranial structures.9 We are exploiting this conservation to examine the molecular mechanisms that mediate programmed murine cranial suture fusion. Although it remains to be proven, we would speculate that mouse and man share similar calvarial molecular specification and sutural biology. The following series of experiments performed in our laboratory illustrate some of the advances in murine cranial suture research and highlight our understanding of the molecular mechanisms governing this system.
THE DURA MATER GUIDES CRANIAL SUTURE FATE
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 murine model of cranial suture fusion
First, we defined the temporal sequence of cranial suture fusion in our models. By serially sectioning murine calvaria, our laboratory and others have demonstrated that the posterior frontal (PF) suture fuses in an anterior to posterior and endocranial to ectocranial direction from postnatal days 12-22 in the rat and 25-45 in the mouse.10,11,12 We feel that this PF suture is analogous to the human metopic suture. In addition, we have demonstrated that all other cranial sutures, including the coronal (COR) and sagittal (SAG), remain patent for the life of the animal. The disparate fate of these sutures was opportune because it enabled us to compare and contrast gene and protein expression in fusing and patent sutures.
Second, we developed an
Finally, using loupe magnification and micro-dissection, we established enriched cranial suture-associated dural and neonatal calvarial osteoblast cell lines.16,17,18 These cell lines enabled us to understand the dura mater-derived signals and their effects on osteoblast phenotype.
In 1996, Roth
We were surprised by these data because they suggested that the dura mater played an essential role in guiding cranial suture fate. Furthermore, we hypothesized that the PF dura mater was secreting soluble factors that were prevented from diffusing into the overlying cranial suture by the impermeable silicone membrane. This lead us to explore programmed regional specialization of the PF vs. SAG dura mater.
In order to investigate the regional specialization of the dura mater, Levine
In order to explore the regional difference in dural cells, we isolated the PF and SAG sutures of Sprague-Dawley rats.16 The underlying suture-associated dura mater was dissected free of the overlying suture complex and individual PF and SAG dural cell lines were established. First-passage SAG suture-derived dural cells demonstrated decreased cellular contact inhibition and significantly increased rates of cellular proliferation when compared to PF dural cells. In contrast, PF dural cells expressed more than twice as much alkaline phosphatase activity and collagen I protein. The PF and SAG dural cells both possessed the capacity to form bone nodules. Collectively, these data demonstrated that phenotypic differences exist between early-passage dural cells derived from fusing and patent sutures. The formation of bone nodules suggests that both PF and SAG dura mater contain a population of osteoblast-like cells; however, elevated collagen I protein expression and alkaline phosphatase activity in PF dural cells suggest that the PF dura may contain more mature osteoblast-like cells. Cellular maturation and differentiation of PF dural osteoblast-like cells may be responsible for decreased cellular proliferation and enhanced contact inhibition.
The differences identified in suture-specific dural cells, in conjunction with the rotational and translocational cranial suture data, supported the hypothesis that the murine dura mater was regionally differentiated and provided paracrine signals to the overlying murine suture complex. Furthermore, the increase alkaline phosphatase activity and bone nodule formation in the PF dura suggested that this tissue contained a sub-population of osteoblastic cells that was markedly attenuated in SAG dura. Although it remains to be proven, we hypothesized that this sub-population of osteoblastic cells was contributing to PF suture fusion.
The pericranium and cranial suture mesenchyme do not control cranial suture fate
Moss was the first to investigate the role of the pericranium.21 Stripping the pericranium from neonatal rat calvaria, he observed normal PF suture fusion and COR suture patency. Opperman
By analyzing the gene expression within the pre-fusing, isolated cranial suture complex, Spector
Taken together, these experiments suggested that the osteogenic machinery within the isolated cranial suture complex remained primed awaiting osteoinductive paracrine signals from the underlying dura mater. These results lead us to investigate the nature of the dura mater-derived paracrine signals in the following series of experiments.
GROWTH FACTOR EXPRESSION IN CRANIAL SUTURE BIOLOGY
While the dura mater, independent of cranial base forces, appeared critical in determining sutural fate, the precise mechanisms mediating the dura mater-suture interaction remained unknown. In order to investigate dura-suture cytokine communication, we used a candidate gene approach. By
The insulin-like growth factors (IGF-I and IGF-II) are 7.6 and 7.5 kD dimeric peptides, respectively.24, 25 Both IGF-I and IGF-II are involved in bone formation and repair.26,27,28,29 For example, The IGFs exert their mitogenic effects and induce collagen synthesis in osteoblasts through IGF type I and II receptors. Numerous studies have demonstrated that IGF I and IGF-II enhance bone healing when injected locally or even administered systemically.30,31,32,33,34,35 Interestingly, Canalis and Lian demonstrated that IGF-I and IGF-II stimulate osteoblasts to express osteocalcin.36 Since osteocalcin is expressed only by mature osteoblasts, the authors hypothesized that IGFs drive osteoblast differentiation.
In order to determine if IGF-I and IGF-II played a role in PF suture fusion, Bradley
The transforming growth factor beta (TGF-() superfamily includes a number of important growth factors including three TGF-( isoforms, the bone morphogenetic proteins, activins, inhibins, and growth and differentiation factors. TGF-(1, -(2, and -(3 are three closely related isoforms that are widely expressed during skeletal morphogenesis and bone repair.38,39,40,41 These TGF-?s stimulate osteoblast proliferation and induce the synthesis of collagen, osteocalcin and other extracellular matrix proteins.42,43,44,45,46 In addition, TGF-?s enhance extracellular matrix depostition by inhibiting osteoclast activity and down-regulating the expression of tissue metalloproteinases.47,48,49,50,51 Furthermore, exogenous TGF-?1 enhances bone deposition and the healing of bone defects.52,51,52,53,54,55
In 1997, Roth
Taken together, the human craniosynostotic findings and murine data implicate TGF-? signaling in the regulation of cranial suture fate. In addition, the similarities in TGF-? isotype expression support the supposition that programmed suture fusion in murine models and premature fusion in man share, at least in part, evolutionarily conserved signaling pathways. Additional work is necessary to clarify the complex roles of TGF-?s in cranial suture biology.
The FGFs are a large family of at least 19 cytokines that regulate cell migration, angiogenesis, bone development and repair, and epithelial-mesenchymal interactions.62,63,64,65,66 FGF-2 is the most abundant ligand and it has been shown to stimulate osteoblast proliferation and enhance bone formation
Most and Mehrara
Since gain-of-function FGF receptor (FGFR) mutations are the most common syndromic cause of craniosynostosis, our laboratory and others have investigated the expression of FGF receptors in the murine model. Mehrara
Taken together, these studies suggested that FGF signaling played an important role in cranial suture biology.
Since accumulating evidence suggested that FGFs critically regulated cranial suture physiology, we attempted to determine if we could reverse programmed cranial suture fate by manipulating FGF-biologic activity. In order to do this, Greenwald
Normal cranial suture biology in murine models is very complex and seems to require a coordinated cascade of molecular signals from the underlying dura mater. 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. Ultimately, by understanding the mechanisms that mediate murine cranial suture biology, we may someday intelligently develop targeted biologically based strategies to treat or reverse prematurely fusing sutures.