Transforming growth factor-beta 2 and TGF-beta 3 regulate fetal rat cranial suture morphogenesis by regulating rates of cell proliferation and apoptosis

Cranial vault sutures are the major intramembranous bone growth sites during rapid expansion of the neurocranium. To function as bone growth sites, sutures need to remain patent, while allowing rapid bone formation at the edges of the bone fronts. Premature osseous obliteration of sutures (craniosynostosis) by fusion of bone fronts across the suture site prevents further bone formation at this site, often leading to severe facial dysmorphology. Although several growth factor receptor and transcription factor mutations have been implicated in craniosynostosis, the underlying mechanisms leading to sutural obliteration remain unclear. Previous studies have shown that dura secreted soluble factors responsible for maintaining suture patency and that suture fusion observed in the absence of dura was preceded by elevated levels of DNA synthesis and collagen production in the suture region. The use of neutralizing antibodies in a fetal calvarial culture model further demonstrated that removal of transforming growth factor (TGF) -beta 3 activity induced premature sutural obliteration, whereas removal of TGF-beta 2 activity prevented sutural obliteration. Data presented here demonstrate that suture obliteration induced by removal of TGF-beta 3 activity was preceded by elevated levels of DNA synthesis, similar to that seen upon removal of the dura. Addition of exogenous TGF-beta 3 to calvaria cultured without dura both prevented suture obliteration and reduced DNA synthesis to levels comparable to those seen with intact dura. Addition of exogenous TGF-beta 2 to calvarial cultures induced sutural fusion accompanied by elevated levels of cell proliferation. However, sutures rescued from obliteration by removal of TGF-beta 2 activity did not have decreased levels of cell proliferation, but rather appeared to be due to inhibited differentiation. In all calvaria in which sutures remained patent in culture, numbers of apoptotic cells were high within the suture, whereas in sutures destined to fuse, numbers of apoptotic cells were low. Results indicate that one of the critical regulators of suture patency is cell number. Alterations in cell number can trigger premature differentiation of cells, resulting in sutural obliteration. Furthermore, a complex interplay between closely related molecules is required to maintain cranial vault sutures in an unossified state, while allowing new bone to be formed at the edges of the bone fronts.