The Role of BMP Signaling in Mediating Stimulatory Effects of FGF-FGFR Signaling on Odontoblast Differentiation in Dental Pulp

Odontoblast differentiation is dependent on multiple signaling molecules, including growth factors stored in the extracellular dentin matrix. Our previous studies have demonstrated that Fibroblast Growth Factor 2 (FGF2) exerted stage-specific effects on odontoblast differentiation in primary dental pulp cultures. FGF2 stimulated differentiation of functional odontoblasts expressing high levels of Dmp1 from early progenitors but inhibited the terminal differentiation of functional odontoblasts to fully differentiated odontoblasts expressing high levels of Dspp. These stimulatory effects involved activation of FGFR and ERK1/2, and increased levels of expression of Bmp2, Runx2, and Osx (components of BMP signaling) suggesting the involvement of BMP signaling in FGF2-induced Dspp expression. Therefore, the overall goal of the studies outlined in this thesis was to examine the role of BMP signaling in stimulatory effects of FGF signaling on odontoblast differentiation of dental pulp cells. We showed that BMP2 did not affect the extent of mineralization but rapidly (within ~12-24 hrs) stimulated expression of Dspp and intensity of DSPP-Cerulean transgene in a concentration-dependent manner without affecting the percentage of DSPP-Cerulean+ odontoblasts. In contrast to Dspp, BMP2 rapidly (within ~24-48 hrs) decreased expression of Dmp1, Bsp, DMP1-mCherry, and BSP-GFP in a concentration-dependent manner. Inhibition of the BMP and FGF signaling pathways by noggin and SU5402, respectively, did not have marked effects on the extent of mineralization but decreased expression of Dspp stimulated by these growth factors. These inhibitory effects were long-lasting and observed up to 14 days after removal of the growth factors. Additional experiments in primary bone marrow stromal cells cultures demonstrated that early and limited exposure to BMP2 did not affect mineralization but decreased expression of Dmp1, Bsp, and respective transgenes. Taken together, our results demonstrated that activation of the canonical BMP signaling pathway during the proliferation phase of in vitro growth resulted in significant increases in the expression of Dspp, and these increases were mediated via reciprocal interaction of the BMP and FGF signaling pathways. These data will further elucidate the perspectives of using both FGF2 and BMP2 in dentin regeneration applications

Insight into Stage-Specific Effects of Fibroblast Growth Factor Signaling on Dental Pulp Cells

Odontoblast differentiation and reparative dentinogenesis are dependent on multiple signaling molecules, including members of Fibroblast Growth Factor (FGF) family. Previous studies have demonstrated both positive and negative effects of FGFs on odontoblast differentiation in dental pulp. However, the underlying cellular and molecular mechanisms of these opposite effects are unclear. Therefore, the overall goal of the studies outlined in this dissertation was to gain insight into the cellular and molecular mechanisms of the effects of FGF signaling on mineralization and dentinogenesis of pulp cells. To approach this goal, we used dental pulp cultures derived from the coronal portion of unerupted molars from various transgenic mice, which display stage-specific activation of transgenes during odontoblast differentiation. Our observations showed that exposure of progenitors and cells at early stages of odontoblast differentiation stimulated/accelerated their differentiation into functional odontoblasts, expressing high levels of Dmp1 and DMP1-GFP, and low levels of Dspp and DSPP-Cerulean. Additional exposure maintained cells as functional odontoblasts and prevented their differentiation into mature odontoblasts, expressing high levels of Dspp and DSPP-Cerulean and producing mineralized matrix. Withdrawal of FGF2 resulted in complete recovery of mineralization and differentiation of functional odontoblasts into mature odontoblasts. Analysis of FACS-sorted 2.3-GFP– (progenitors) and 2.3-GFP+ (cells at early stages of differentiation) cells confirmed that FGF2 stimulated/accelerated their differentiation into functional odontoblasts, while prevented their differentiation into fully differentiated odontoblasts. Finally, our results showed that stimulatory effects of FGF2 were mediated by activation of the FGFR, MEK/Erk1/2 and BMP/BMPR signaling pathways, whereas inhibitory effects were mediated by re-activation of the FGFR and MEK/Erk1/2 pathways with negative roles in odontoblast differentiation. Taken together, our results demonstrated the stage-specificity of the effects of FGF2 on odontoblast differentiation, suggesting that FGF2 is a viable growth factor for dentin regeneration

Biphasic Effects of FGF2 on Odontoblast Differentiation Involve Changes in the BMP and Wnt Signaling Pathways

Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including Fibroblast Growth Factors (FGFs), Bone Morphogenetic Proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of the components of the BMP (Bmp2, Dlx5, Msx2, Osx) and Wnt (Wnt10a, Wisp2) pathways, and decreased expression of an inhibitor of the Wnt signaling, Nkd2. Further addition of FGF2 during the differentiation/mineralization phase of growth resulted in decreased expression of components of the BMP signaling (Bmp2, Runx2, Osx) and increased expression of inhibitors of the Wnt signaling (Nkd2, Dkk3). This suggests that both BMP and Wnt pathways may be involved in mediating the effects of FGF2 on dental pulp cells

Osteoblast Response to Commercially Available Ti-6Al-4V and Novel Porous Tantalum. In Vitro Biocompatibility Studies

The response of human osteoblasts to materials is crucial for evaluating biocompatibility of an implant material for bone defects. Previous work in our lab demonstrated that the response of human osteoblasts to orthopaedic and dental materials in vitro varies depending on the sex and age of the patient [1]. Osteoblasts from female patients older than 60 years old, adhered less and produced less matrix proteins and calcification than osteoblasts from younger female patients and all ages of male patients. Recently developed, porous tantalum demonstrates improved biomechanical properties for bone and good biocompatibility in in vivo human studies, however there are few, if any, in vitro biocompatibility studies on this material. In this project, we aimed to compare the phenotypic expression of human osteoblasts from young and old female patients to commercially available Ti-6Al-4V and porous tantalum in a well-developed in vitro system. 1. Zhang H, Lewis CG, Aronow MS, Gronowicz G. The effect of patient age on human osteoblasts’ response to Ti-6Al-4V implants in vitro. J. Orthop. Res. 2004;22(1):30-8