Biomolecular mechanisms of calvarial bone induction: Immature versus mature dura mater

The ability of newborns and immature animals to reossify calvarial defects has been well described. This capacity is generally lost in children greate r than 2 years of age and in mature animals. The dura mater has been implic ated as a regulator of calvarial reossification. To date, however, few stud ies have attempted to identify biomolecular differences in the dura mater t hat enable immature, but not mature, dura to induce osteogenesis. The purpo se of these studies was to analyze metabolic characteristics, protein/gene expression, and capacity to form mineralized bone nodules of cells derived from immature and mature dura mater. Transforming growth factor beta-1, bas ic fibroblast growth factor, collagen type I alpha I, osteocalcin, and alka line phosphatase are critical growth factors and extracellular matrix prote ins essential for successful osteogenesis. In this study, we have character ized the proliferation rates of immature (6-day-old rats, n = 40) and matur e (adult rats, n = 10) dura cell cultures. In addition, we analyzed the exp ression of transforming growth factor beta-1, basic fibroblast growth facto r-2, proliferating cell nuclear antigen, and alkaline phosphatase. Our in v itro findings were corroborated with Northern blot analysis of mRNA express ion in total cellular RNA isolated from snap-frozen age-matched dural tissu es (6-day-old rats, n = 60; adult rats, n = 10). Finally, the capacity of c ultured dural cells to form mineralized bone nodules was assessed. We demonstrated that immature dural cells proliferate significantly faster and produce significantly more proliferating cell nuclear antigen than matu re dural cells (p < 0.01). Additionally, immature dural cells produce signi ficantly greater amounts of transforming growth factor beta-1, basic fibrob last growth factor-2, and alkaline phosphatase (p < 0.01). Furthermore, Nor thern blot analysis of RNA isolated from immature and mature dural tissues demonstrated a greater than 9-fold, 8-fold, and 21-fold increase in transfo rming growth factor beta-1, osteocalcin, and collagen I alpha I gene expres sion, respectively, in immature as compared with mature dura mater. Finally , in keeping with their in vivo phenotype, immature dural cells formed larg e calcified bone nodules in vitro, whereas mature dural cells failed to for m bone nodules even with extended culture. These studies suggest that diffe rential expression of growth factors and extracellular matrix molecules may be a critical difference between the osteoinductive capacity of immature a nd mature dura mater. Finally, we believe that the biomolecular bone- and m atrix-inducing phenotype of immature dura mater regulates the ability of yo ung children and immature animals to heal calvarial defects.