Identification of stiffness-induced signalling mechanisms in cells from patent and fused sutures associated with craniosynostosis

Sara Barreto,Dylan J Murray,Arlyng González-Vázquez,Fergal J O’Brien,Andrew R Cameron

doi:10.1038/s41598-017-11801-0

Abstract

Craniosynostosis is a bone developmental disease where premature ossification of the cranial sutures occurs leading to fused sutures. While biomechanical forces have been implicated in craniosynostosis, evidence of the effect of microenvironmental stiffness changes in the osteogenic commitment of cells from the sutures is lacking. Our aim was to identify the differential genetic expression and osteogenic capability between cells from patent and fused sutures of children with craniosynostosis and whether these differences are driven by changes in the stiffness of the microenvironment. Cells from both sutures demonstrated enhanced mineralisation with increasing substrate stiffness showing that stiffness is a stimulus capable of triggering the accelerated osteogenic commitment of the cells from patent to fused stages. The differences in the mechanoresponse of these cells were further investigated with a PCR array showing stiffness-dependent upregulation of genes mediating growth and bone development (TSHZ2, IGF1), involved in the breakdown of extracellular matrix (MMP9), mediating the activation of inflammation (IL1β) and controlling osteogenic differentiation (WIF1, BMP6, NOX1) in cells from fused sutures. In summary, this study indicates that stiffer substrates lead to greater osteogenic commitment and accelerated bone formation, suggesting that stiffening of the extracellular environment may trigger the premature ossification of the sutures.

Highlights

Human calvarial bones are derived from paraxial mesoderm and craniofacial neural crest cells and are mostly formed by intramembranous ossification[1, 2]
Calvarial bone formation and suture development can sometimes be altered in developmental diseases, such as craniosynostosis, which is caused by an acceleration of ossification within patent sutures of the skull, which prematurely fuse restricting brain growth during development
Since bone generation is mediated by signalling mechanisms that include stimuli from the surrounding environment, pathological changes in the physiology of the sutures of children with craniosynostosis may be associated with changes in the stimuli provided by the extracellular environment that impairs the functional capacity of cells within the sutures to sense and respond to these stimuli

Summary

Introduction

Human calvarial bones are derived from paraxial mesoderm and craniofacial neural crest cells and are mostly formed by intramembranous ossification[1, 2]. Calvarial bone formation and suture development can sometimes be altered in developmental diseases, such as craniosynostosis, which is caused by an acceleration of ossification within patent sutures of the skull, which prematurely fuse restricting brain growth during development. Since bone generation is mediated by signalling mechanisms that include stimuli from the surrounding environment, pathological changes in the physiology of the sutures of children with craniosynostosis may be associated with changes in the stimuli provided by the extracellular environment that impairs the functional capacity of cells within the sutures to sense and respond to these stimuli. In order to understand if changes in the substrate stiffness are associated with premature fusion of sutures and the specific mechanotransductive mechanisms that underpin this response, we cultured cells isolated from patent and fused sutures of non-syndromic children diagnosed with craniosynostosis on soft and stiff collagen-coated polyacrylamide substrates. We aimed to elucidate differences in the molecular signalling pathways and in the behaviour of osteoblastic cells from patent and fused sutures and whether these differences are driven by changes in the stiffness of the microenvironment

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