Extrinsic tension results in FGF-2 release, membrane permeability change, and intracellular Ca⁺⁺ increase in immature cranial sutures

There are numerous studies cataloging the temporal profiles of the various growth factors during the morphogenesis of cranial sutures. There are also many clearly documented mutations of the receptors of some of these growth factors such as fibroblast growth factor (FGF)R-2 and FGFR-3 in clinical craniosynostosis. It is obvious, and often concluded, that growth factors play a role or are involved in craniofacial development. However, precisely what that role is, what causes the changes in the growth factor levels, and why these changes occur in the particular temporal and spatial patterns observed remains elusive. Using simple physics, we applied a plasma membrane disruption model and the principles of complex adaptive systems to arrive at a conjecture of calvarial morphogenesis. The purpose of this article is to introduce the concept of complex adaptive systems,to propose our conjecture, and to provide experimental proof of some key steps in this conjecture: tension induces rapid and demonstrable physiological responses in some cells within the immature cranial sutures. These responses include increases of intracellular Ca⁺⁺, plasma membrane permeability, and the release of growth factors, e.g., FGF-2. Paired coronal sutures from 1-week-old Sprague-Dawley rat pups were subjected to either 0.59 N of tensile force or no force for 5 minutes in a protein-free medium. FGF-2 levels in the media were measured by slot blot analysis. Western blot analysis was used to determine FGF-2 levels in the sutures. To determine cell membrane permeability changes, fluorescein-conjugated dextran, with a molecular weight of 10 kd, was added to the media during the 5 minutes with or without tensile force. Laser confocal microscopy was used to compare the amount of entry of this impermeant tracer and the pattern of permeability change at the tissue level. To determine the intracellular pCa⁺⁺, the sutures were first loaded with a calcium indictor, FURA-2 AM, and then subjected isotonically to 0.059 N of tension. The intracellular pCa⁺⁺ was expressed as ratio of Ca⁺⁺-bound FURA-2 to Ca⁺⁺-free FURA-2. The experimental findings were as follows: 1) Sutures, in response to tension, release FGF-2. 2) Sutures contain higher levels of FGF-2 when strained. 3) There is an increase in the sutural cell membrane permeability as a result of tensile strain. 4) The cells along the leading edges of the ossification fronts (at the insertion sites of Sharpey's fibers) demonstrated the maximum permeability increase. 5) There was an immediate (within seconds) increase in intracellular Ca⁺⁺. and 6) This increase in intracellular Ca⁺⁺ caused by tension was reversible and independent of the extracellular Ca⁺⁺ ion availability. In summary, these data support, in part, the conjecture that growth of the brain places strain on the cells within the immature sutures, which causes the iteration of a set of cellular subroutines. These subroutines integrate to generate the emergent property of directed cranial expansion with dissipation of the initiating strains.