Abnormal response to FGF2 in calvarial-derived cells from craniosynostotic rabbits
Michael R. Bykowski1, Darren M. Smith, MD1, James Cray, PhD1, Emily Lensie, BA1, Melissa Smalley, BA1, Christopher Kinsella, MD1, Mark Mooney, PhD1, Phil Campbell, PhD2, Lee Weiss, PhD2, Joseph E. Losee, MD1, Gregory M. Cooper, PhD1.
1University of Pittsburgh, Pittsburgh, PA, USA, 2Carnegie Mellon University, Pittsburgh, PA, USA
Background: Craniosynostosis (CS) is a pathological condition defined as the premature fusion of the sutures of the skull. The birth prevalence of CS is estimated to be 300-500 cases per 1,000,000 live births. CS involves the overgrowth of bone at the osteogenic fronts of the developing calvarial bone. Normally, where osteogenic fronts meet each other, a region of tissue between the fronts remains undifferentiated. This region of non-bony tissue (the suture) allows for prenatal and postnatal brain growth. In CS, bone growth is accelerated at the osteogenic fronts, and the region between the fronts differentiates into bone and obliterates the suture, leading to a bone-filled joint between osteogenic fronts (sysnostosis). Our goal is to investigate the role of Fibroblast Growth Factor-2 (FGF2) in cellular proliferation of calvarial bone-derived cells from wild-type or craniosynostotic rabbits.
Methods:Calvarial bone was harvested from 10-day old wild-type New Zealand White rabbits (n=7) or rabbits with prenatal coronal suture synostosis (n=4). Bone-derived cells (suture- and non-suture-associated bone) were stimulated with various concentrations of FGF2 - either with soluble FGF2 or ‘solid-phase’ bio-printed FGF2. Solid-phase FGF2 was immobilized using inkjet-based deposition onto fibrin-coated glass slides, which were then seeded with calvarial-derived bone cells and cultured with standard medium. Cell proliferation was assessed 2 days after seeding through MTS assay (for soluble FGF2 experiments) or through phase-contrast imaging (for solid-phase bio-printed FGF2 stimulation).
Results: Qualitative image analysis of cells treated with solid-phase FGF2 suggests craniosynostotic suture bone-derived cells to be less affected by FGF2 compared to wild-type-derived bone cells; the latter demonstrated a marked increase in cell proliferation. MTS assays revealed wild-type non-suture bone-derived cells proliferated significantly more in response to FGF2 compared to craniosynostotic non-suture bone-derived cells (p<0.05). In general, we found that non-suture bone-derived cells have the greatest cell proliferation potential (p<0.001). Moreover, proliferation of craniosynostotic suture bone-derived cells occurs biphasically in response to FGF2 - i.e., proliferation peaks at 1 ng/ml and is inhibited at 10 ng/ml FGF2.
Conclusion: Proper morphogenesis and growth of the calvarial vault is dependent on the balance between osteogenesis of calvarial bones and cellular proliferation at the cranial sutures. While the unstimulated proliferation rate of craniosynostotic calvarial bone-derived cells was higher than their wild-type counterparts, they were less responsive than wild-type cells to exogenous FGF2. These effects may be mediated through elevated FGF2 signaling inherent to craniosynostotic calvarial-derived cells. These findings suggest that the strength of FGF2 signaling and/or concurrent calvarial cell responses regulates cellular proliferation of calvarial bone and suture. Furthermore, these findings - in addition to the well-known role of FGF signaling in normal calvarial vault osteogenesis - implicate aberrant FGF signaling to play a role in tilting the balance of osteogenesis and suture cell proliferation toward craniosynostosis in our rabbit model.
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