Gene Analysis and Osteoblast Phenotype Characterization of Non-Syndromic Metopic Craniosynostosis

Objective: Craniosynostosis results in restricted skull growth and increased intracranial pressure, and are non-syndromic in 60% of all cases. Metopic synostosis, which results in trigonocephaly, is one of the most common single suture synostosis, but the molecular mechanisms are poorly understood. In this study, we characterized osteogenic profile and paracrine factor production in cells isolated from normal bone distal from sutures (NB), patent suture (PAT) and fused suture (FUS).

Method: Craniosysnostosis cells (CSC) from NB, PAT, and FUS were isolated from six patients undergoing corrective surgery for metopic craniosynostosis. Osteoblast phenotype was characterized by alkaline phosphatase (ALP) and osteocalcin production. Expression of integrins and Wnt and BMP pathway molecules was measured by real-time qPCR. To determine the effect of CSC on surrounding MSCs, CSC were co-cultured with HMSCs. After seven days, the CSC were removed and cell number, ALP, and secreted factors analyzed in the HMSCs. Data are mean±SEM (n=6/condition, ANOVA and post-hoc Bonferroni’s Student's t-test).

Result: ALP and osteocalcin were higher in cultures of CSC from FUS than from NB or PAT. Expression of DKK1 was higher and β-catenin lower in FUS CSC than in NB and PAT CSCs, indicating reduced canonical Wnt signaling in FUS sutures. BMP4 levels were higher in FUS CSC than PAT or NB. ITGA5 and ITGB1 expression was higher in PAT CSCs in comparison with NB or FUS. MSCs co-cultured with FUS CSC had higher ALP, and secreted more osteocalcin, osteoprotegerin, VEGF, TGFβ1, BMP2, and BMP4 than MSCs cultured with NB or PAT CSC.

Conclusion: Cells isolated from fused sutures in non-syndromic craniosynostosis present a more differentiated osteogenic phenotype. Cells from fused sutures produce an osteogenic environment that induces osteoblastic differentiation in adjacent MSCs. Gene expression analysis suggests that BMP4, ITGA5, ITGB1, and DKK1 may play an important role in metopic suture fate.