Date of Award


Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

First Advisor

John Persing


Craniosynostosis is the premature fusion of one or more of the cranial sutures resulting in skull deformity and possible brain dysfunction. It occurs in roughly 1 in 2,000 live births.1 It may be associated with syndromes or occur sporadically. Any cranial suture may be involved. The descriptions of the skull dysmorphologies have led to numerous hypotheses regarding the developmental trajectory of the synostosed skull, including the role of the cranial vault and cranial base. As proposed originally by Virchow (1851),2 the shape of the skull in craniosynostosis is usually attributed to a lack of local growth perpendicular to the fused suture with compensatory growth occurring at adjacent patent sutures. This change in growth vectors is a variation on the highly coordinated adjustment required of the normally developing head. The reasons these growth vectors change may be directly related to changes in applied stress. However, the question still remains as to why the suture fuses prematurely. The cause of premature fusion of cranial sutures has been speculated to be either due to physical constraint (i.e. stress)3,4 or to genetic mutation.5-8 Although some genetic mutations have been identified in individuals with craniosynostosis, the role of these mutations in pathways regulating suture patency and/or skull growth has not been characterized. To date, only coronal suture craniosynostosis has been found to be associated with a specific genetic mutation. Even still screens of non-syndromic patients with coronal craniosynostosis have found varied expression of this mutation, P250R a fibroblast growth factor receptor (FGFR) 3.9-12 In fact, only 50% of isolated cases of coronal synostosis have been shown to carry the mutation.9-12 Current consensus is that the FGFR3 mutation causes a particular syndrome (Muenke syndrome) with variable expressivity and incomplete penetrance.11-12 As for the remaining majority of craniosynostosis cases no consensus exists. The influence of physical constraint, or stress, is currently poorly understood. Despite studies suggesting that in utero constraint leads to craniosynostosis,13,3,4 other studies concluded that constraint leads only to deformation of the skull while sutures remain patent.14,15 Thus, we have a very limited understanding of the relationship between physical stress to suture fusion. Genetic mutation and/or physical stress may play a role in causing premature suture fusion, but neither can affect the ontogenetic pathway of skull and sutures without having an impact on the entire craniofacial system. The osseous elements of the skull do not develop in isolation; rather the post-natal skull, brain, and dura mater develop in intimate physical and biochemical contact with one another. The precise nature of the interactions is unclear. Many studies have demonstrated that the presence of dura mater is necessary to maintain suture patency, and further, that the signal mediating suture fusion involves soluble factors, rather than biomechanical factors or cell-cell interactions.16-18 Additionally, studies have hypothesized that complex cell signaling from dura to osteogenic cell populations is responsible for patency of the suture.16-20 However, the biomechanical/biochemical mechanisms necessary for production of cranial vault phenotypes in craniosynostosis are not elucidated by these findings. A functional approach to the study of skull form was introduced by van der Klaauw (1948-1952)21 and expanded on by Moss and colleagues.22,23 In particular, Moss and Young (1960)22 presented a functional analysis to neurocranial growth, proposing that the size and shape of the cranial vault is determined by the form and orientation of the dura mater, which in turn is a direct reflection of the form of the brain. Citing Popa (1936),24 Moss and Young (1960)22 point out that the brain is encapsulated by the dura mater, which is firmly attached to the chondrocranium from its initiation. Since the dura mater and skull base are so firmly integrated at specific sites, a system of forces is produced by the growing brain, placing pressure against this capsule formed by the dura and skull tissues surrounding the brain. The dural folds produced by these attachments sites underlie the calvarial sutures and this relationship is proposed as playing a part in normal suture closure. Moss and Young (1960)22 suggested biomechanical forces produced by growth of the brain as the means of communication between adjacent tissues. The role of biomechanical forces in signaling diffusion of growth factors in communication among tissues has been supported experimentally,25-31 suggesting both biochemical and biomechanical influences on the craniofacial phenotype. Whatever the mechanism for communication we know that a change in the growth trajectory of one of these tissue units influences changes in the trajectory of the others. For example, mechanical forces acting on the external neurocranium, such as binding of immature heads32,33 or a habitual sleeping position,34,35 changes the shape of the endocranium and neural mass. In fact, Babler and Persing et al. demonstrated that suture fusion shortly after birth via application of adhesive to the sagittal suture of rabbits causes both deformation of the basicranial and facial dimensions.36 Likewise, changes in arrangement of dural attachment sites by way of cranial base deformation (experimentally or naturally produced) alter the shape of the outer skull and the neural mass.37,38 So, too changes in brain volume such as hydrocephalus, anencephaly, and microcephaly result in adjustments in neurocranial shape.39-42 In summary, one of the primary goals in the study of craniosynostosis is to determine the cause of premature suture fusion and its relationship to observed craniofacial dysmorphology. Beyond understanding the genetic mechanisms potentially underlying premature suture fusion, determination of the cause of craniosynostosis requires knowledge of the development of the entire craniofacial complex prior to, during, and following suture fusion. By the time children are diagnosed with craniosynostosis, the suture has already fused and the associated dysmorphology is well established. Thus, the data required to test directly hypotheses related to the cause of suture fusing is not available in humans and must be sought in animal models. Studies of human data are constrained to the more modest goal of acquiring a quantitative depiction of the phenotypes associated with suture fusion. Within this context, morphology and growth can be evaluated in individuals with craniosynostosis and the findings compared to perform clearer hypotheses to be tested in the appropriate animal models. In this report, a new in vitro model (Microdistractor) is defined (Chapter 3) wherein a linear stress can be applied to a system. Our data suggests the Microdistractor device as effective for studying the cellular response to distraction stresses. As such a murine suture is stressed in this system and the histologic and gene expression changes are noted (Chapter 1). The application of oscillatory stress to cranial sutures results in fusion of both the posterior frontal and the normally patent sagittal suture. However, distractile stress did not cause fusion. This later finding is a likely result of the existence of a range of acceptable stresses. Thus, the stress applied to the suture in distraction caused the two calvarial halves to undergo too great of separation for bony bridging to occur. Both stressed groups however, did demonstrate the same gene expression relative to control: significantly increased expression of the bone differentiation markers Runx2 and the late marker AP with nearly no expression of Noggin, a bone inhibitor. Thus, mechanical stress influenced the cells involved in sutural fusion and stimulated them to undergo osteogenic differentiation. These findings were then compared with an animal (rabbit) model that spontaneously develops craniosynostosis in utero (Chapter 2). Our results suggest that pathologic rabbit coronal sutures progress toward complete suture fusion in vitro. Furthermore, the expression patterns of Noggin, Runx-2, and AP for a fusing suture paralleled that of our stressed model (Chaper 1). Thus, Noggin expression was decreased and Runx-2 and AP were increased in craniosynostosis. Finally, pre-osteoblasts were biomechanically stressed within a collagen gel using the Microdistractor model (Chapter 4). Proliferative changes and genes of osteogenic differentiation were monitored. Cells undergoing linear distraction experienced rapid proliferation with a delayed expression of markers of osteogenic differentiation; whereas, cells undergoing oscillation had a rapid expression of osteogenic markers, but a cellular proliferation pattern indistinguishable from that of unstressed controls. These findings may help to explain the factors that occur in patients with craniosynostosis. For instance if a constant stress similar to distraction were to be applied a proliferative response would occur, when the stress is removed or oscillated the proliferated populations of cells may osteodifferentiate and lead to fusion. At the end of this series we conclude that stress induces the same gene expression patterns as craniosynostosis and the particular pattern of stress application is crucial in determining the cellular response.