Ephrin-B stimulation of calvarial bone formation.

INTRODUCTION: Ephrin-B2 on osteoclasts was reported to promote bone formation as part of homeostasis by activating the EphB4 tyrosine kinase receptor on osteoblasts. Little is known about the role of ephrin-B signaling to EphBs in developmental bone formation. RESULTS: We observed expression of an ephrin-B2 LacZ chimeric allele in the periosteum, sutural bone fronts, and dura mater of embryonic and neonatal mice. Expression in the adult skull was confined to sutures, but was heavily upregulated at sites of bone injury. Culture of embryonic calvariae with soluble recombinant ephrin-B2/Fc doubled their bone content without altering suture width or overall skull morphology. Ephrin-B2/Fc also stimulated osteoblast marker gene expression in cultured MC3T3 preosteoblastic cells without the need for type 1 collagen-induced differentiation. EphB4 was absent in embryonic and adult skulls. However, EphB1 and EphB2, both physiological receptors for ephrin-Bs, were expressed at sites of osteogenesis, and EphB1 knockout mice displayed a reduction in calvarial bone content compared to controls. CONCLUSIONS: These data support a role for ephrin-B2 in the development and healing of bone through activation of osteoblast-specific gene expression. EphB1 and EphB2 are likely candidates receptors for the ephrin-B2 in bone.

Timing of Egf treatment differentially affects Tgf-beta2 induced cranial suture closure.

Premature suture obliteration results in an inability of cranial and facial bones to grow, with craniofacial dysmorphology requiring surgical correction as a consequence. Understanding signaling pathways associated with suture morphogenesis might enable non-invasive treatment of patients with fused sutures. Tgf-beta 2 induces premature suture fusion associated with increased cell proliferation both in vitro and in vivo. Tgf-beta 2 and Egf signal transduction pathways use some signaling proteins in common to regulate proliferation and differentiation, leading to speculation that these two pathways converge to regulate normal suture development. It was therefore hypothesized that Egf could induce suture fusion, and that Tgf-beta 2-induced suture closure occurred via an Egf-dependent pathway. A well-established fetal calvarial organ culture system was used to expose developing E19.5 fetal rat coronal sutures to Egf, Tgf-beta 2 and SC-120, a blocker of Egf receptor activity. Co-culture experiments examined the effect of Egf on Tgf-beta 2-induced suture closure when Egf was given either prior to or after Tgf-beta 2 treatment. Histomorphometric measurement of suture width was done on sagittal sections through coronal sutures harvested after 5 days in culture. Western blotting using phospho-antibodies against Egf receptors was used to confirm Egf receptor activity. Suture width increased with increasing concentrations of Egf, demonstrating that Egf-induced cell activity alone was not sufficient to cause premature suture obliteration. Egf administered prior to Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced suture obliteration, demonstrating that pre-exposure of cells to this powerful mitogen prevented their response to signals induced by Tgf-beta 2. However, Egf added after Tgf-beta 2 treatment had no effect on Tgf-beta 2-induced suture closure. Blocking Egf activity after Tgf-beta 2 treatment rescued sutures from Tgf-beta 2-induced obliteration, indicating that Tgf-beta 2 required Egf activity to induce suture obliteration. Appropriate timing of signal generation by Egf and Tgf-beta 2 is critical for normal suture development and maintenance of suture patency.

Biocompatibility and osteogenic potential of new generation endodontic materials established by using primary osteoblasts.

INTRODUCTION: Generex A and Generex B (calcium silicate based), Capasio (calcium-phospho-alumino silicate based) along with Ceramicrete-D (magnesium phosphate based) are being introduced as a new generation of endodontic materials with the potential to facilitate bone healing. The aim of this study was to evaluate the biocompatibility and osteogenic potential of these new materials by using primary osteoblasts. METHODS: Primary osteoblasts were prepared from rat calvaria and exposed to mineral trioxide aggregate (MTA), Generex A, Generex B, Capasio, and Ceramicrete-D prepared to standardized size and shape (n = 5). Trypan blue staining was used to evaluate cell viability from 1-6 days. Mineralization potential was evaluated by scanning electron microscopy for the presence of mineralized nodules. Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests. RESULTS: Only Generex A and MTA allowed cell growth and proliferation throughout the experiment. There were statistically significant differences between groups throughout the experiment beginning on day 1. The greatest amount of cell growth was consistently observed with Generex A and MTA. There was no difference in mineralized nodule formation between any test materials. CONCLUSIONS: Generex A was the only new generation endodontic material that supported primary osteoblast growth; no material besides MTA facilitated nodule formation.

Erk1/2 signaling is required for Tgf-beta 2-induced suture closure.

Transforming growth factors beta (Tgf-betas) act by means of Smad signaling pathways and may also interact with the mitogen-activated protein kinase pathway. The hypothesis was tested that Erk1/2 signaling is required for Tgf-beta2-induced suture closure, by culturing embryonic mouse calvariae in the presence of Tgf-beta2 with or without Erk1/2 inhibitor PD98059 (PD). Suture widths were measured daily, and microdissected sutures and bones were homogenized and protein analyzed by Western blots. Tgf-beta2 induced narrowing of the sutures after 72 hr, an effect inhibited by treatment with PD. Erk1/2 and Egf but not Smad2/3 protein expression was up-regulated by Tgf-beta2 calvarial tissues at 72 hr. PD inhibited endogenous and Tgf-beta2-stimulated Erk1/2 protein as well as Tgf-beta2-stimulated Egf, but increased Smad2/3 protein expression. In tissues harvested 0, 15, and 30 min after exposure to Tgf-beta2, Erk1/2 phosphorylation was up-regulated after 15 min, an effect abrogated by the simultaneous addition of PD. In summary, Tgf-beta2 stimulated Erk1/2 phosphorylation and induced Egf and Erk1/2 expression, associated with suture closure after 72 hr. Blocking Erk1/2 activity with PD inhibited these effects but increased Smad2/3 expression. We postulate that Tgf-beta2 regulates suture closure directly by means of phosphorylation of Erk1/2 and indirectly by up-regulating Erk1/2, a substrate for Fgf receptor signaling required for Fgf induction of premature suture obliteration.