Studies in Cranial Suture Biology: In Vitro Cranial Suture Fusion

Abstract

The biology underlying craniosynostosis remains unknown. Previous studies have shown that the underlying dura mater, not the suture itself, signals a suture to fuse. The purpose of this study was to develop an in vitro model for cranial-suture fusion that would still allow for suture-dura interaction, but without the influence of tensional forces transmitted from the cranial base. This was accomplished by demonstrating that the posterior frontal mouse cranial suture, known to be the only cranial suture that fuses in vivo, fuses when plated with its dura in an organ-culture system. In such an organ-culture system, the sutures are free from both the influence of dural forces transmitted from the cranial base and from hormonal influences only available in a perfused system. For the cranial-suture fusion in vitro model study, the sagittal sutures (controls that remain patent in vivo) and posterior frontal sutures (that fuse in vivo) with the underlying dura were excised from 24-day-old euthanized mice, cut into 5 × 4 × 2-mm specimens, and cultured in a chemically defined, serum-free media. One hundred sutures were harvested at the day of sacrifice, then every 2 days thereafter until 30 days in culture, stained with H & E, and analyzed. A subsequent cranial-suture without dura in vitro study was performed in a similar fashion to the first study, but only the calvariae with the posterior frontal or sagittal sutures (without the underlying dura) were cultured. Results from the cranial-suture fusion in vitro model study showed that all sagittal sutures placed in organ culture with the underlying dura remained patent. More importantly, the posterior frontal sutures with the underlying dura, which were plated-down as patent at 24 days of age, demonstrated fusion after various growth periods in organ culture. In vitro posterior frontal mouse-suture fusion occurred in an anterior-to-posterior direction but in a delayed fashion, 4 to 7 days later than in vivo posterior frontal mouse-suture fusion. In contrast, the subsequent cranial-suture without dura in vitro study showed patency of all sutures, including the posterior frontal suture. These data from in vitro experiments indicate that: (1) mouse calvariae, sutures, and the underlying dura survive and grow in organ-culture systems for 30 days; (2) the local dura, free from external influences transmitted from the cranial base and hormones from distant sites, influences the cells of its overlying suture to cause fusion; and (3) without dura influence, all in vitro cranial sutures remained patent. By first identifying the factors involved in dural-suture signaling and then regulating these factors and their receptors, the biologic basis of suture fusion and craniosynostosis may be unraveled and used in the future to manipulate pathologic (premature) suture fusion.