"Introduction: Craniosynostosis (CS) is a bone developmental condition that affects 1 in 2100 children worldwide, characterised by premature ossification of the cranial sutures. Particularly, non-syndromic-CS (NS-CS) has been associated to microenvironmental causes. However, little is known about the signalling pathways that govern this skull suture premature ossification. Thus, we hypothesize that by investigating the role of microenvironmental cues in NS-CS, we can identify novel ossification therapeutic targets that could be utilised to develop novel biomaterials-based therapeutic treatments for bone fracture healing in adults.
Methodology: Cells were isolated from patent (unfused) sutures, fused sutures and calvarial bone of children (5-28 months) diagnosed with NS-CS. Tissues were collected during cranial vault remodelling –standard CS surgical procedure- at CHI at Temple Street after parental consent and ethical approval were obtained. To evaluate their osteogenic potential, alkaline phosphatase (ALP) activity and extracellular matrix mineralization of cells cultured for 7-21 days in growth (GM) and osteogenic medium (OM) were quantified. Subsequently, to understand how variations in the substrate stiffness affect premature ossification, cells were cultured on soft (10 kPa) and stiff (300 kPa) collagen-coated polyacrylamide substrates. Then, their osteogenic potential and morphological responses were evaluated. The differences in the mechanoresponse of these cells were further investigated with a 96 gene PCR array to identify potential therapeutic targets.
Results: Cells from patent and fused sutures expressed similar ALP activity and extracellular matrix mineralisation at the different evaluated time-points, when cultured with GM. Interestingly, when cultured with OM, cells from fused sutures expressed higher mineralisation levels and ALP activity. Thus, suggesting that cells from fused sutures have a stronger osteogenic response than cells from patent sutures when biochemically stimulated. Furthermore, when cultured with GM on soft and stiff substrates, cells from both patent and fused sutures exhibited morphological changes and increase in their spreading area, in a stiffness-increasing manner. Particularly, cells from fused sutures showed a bigger and rounded shape, resembling osteoblasts while cells from patent sutures were elongated, resembling mesenchymal stem cells. Finally, when combining variations in the substrate stiffness and OM, a stiffness-dependent upregulation of genes mediating bone development (TSHZ2, IGF1), activation of inflammation (IL1β), involved in the breakdown of extracellular matrix (MMP9) and controlling osteogenic differentiation (WIF1, BMP6, NOX1), was observed in cells from fused sutures. These findings suggest that the increased osteogenic potential of cells from fused sutures might be associated to the activation of the BMP6, IGF1 and/or MAPK-associated non-canonical WNT pathways.
Conclusions: Our results further suggest that NS-CS may be linked to an abnormal mechanical environment. Understanding the changes in the regulation of genes associated with the premature suture ossification in CS opens up avenues to not only understand better this developmental condition but also will help us to design novel therapeutic strategies to accelerate non-union bone fracture healing in adults.
1. Barreto, S. et al., Sci. Rep. 7, 11494 (2017).
This work was funded in part by the Children's Health Foundation Temple Street (RPAC-2013-06 and RPAC-19-01), and by the European Research Council (ERC Advanced Grant ReCaP project #788753)."