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Porous tantalum (Ta) implants have been successful in various orthopedic procedures for patients with compromised boneforming abilities. Previous studies demonstrated that human osteoblast (HOB) cultures from older female patients produced less bone on implant materials in vitro compared to HOBs from age-matched male and younger female patients. In this study, the responses of HOBs from younger (<45) and older (>60 years old) female patients were compared on Ta, titanium fiber mesh (TFM) and tissue culture plastic. Adhesion, proliferation, and mineralization were greater in cells from younger patients than from older patients. Cell adhesion was slightly higher on Ta than TFM or plastic. However, Ta highly stimulated cell proliferation with a 4-and 6-fold increase compared to TFM for cells from younger and older patients, respectively, and 12-and 16-fold increase in proliferation compared to cells on plastic ( p = 0.001). At 3 weeks, mineralization was significantly higher on Ta compared to TFM for HOBs from older patients ( p = 0.05). Expression levels of bone matrix markers demonstrated differences dependent on age and substrate. Scanning electron micrographs revealed HOBs covering the surfaces and entering the pores of both Ta and TFM. In conclusion, tantalum greatly stimulates cell proliferation, and improves the ability of HOBs from older patients to form bone. ß

Members of the fibroblast growth factor (FGF) family play essential and important roles in primary and reparative dentinogenesis. Although there appears to be a general agreement on the effects of FGF signaling on the proliferation of pulp cells, there are conflicting results regarding its effects on odontoblast differentiation. We recently examined the effects of continuous exposure of dental pulp cells to FGF2 and showed that the effects of FGF2 on differentiation of progenitor cells into odontoblasts were stage specific and dependent on the stage of cell maturity. The purpose of this study was to gain further insight into cellular and molecular mechanisms regulating the stimulatory effects of FGF2 on odontoblast differentiation. To do so, we examined the effects of early and limited exposure of pulp cells from a series of green fluorescent protein (GFP) reporter transgenic mice that display stage-specific activation of transgenes during odontoblast differentiation to FGF2. Our results showed that early and limited exposure of pulp cells to FGF2 did not have significant effects on the extent of mineralization but induced significant increases in the expression of Dmp1 and Dspp and the number of DMP1-GFP + and DSPP-Cerulean + odontoblasts. Our results also showed that the stimulatory effects of FGF2 on odontoblast differentiation were mediated through FGFR/MEK/Erk1/2 signaling, increases in Bmp2, and activation of the BMP/BMPR signaling pathway. These observations show that early and limited exposure of pulp cells to FGF2 alone promotes odontoblast differentiation and provides critical insight for applications of FGF2 in dentin regeneration.

Dentinogenesis is a complex and multistep process, which is regulated by various growth factors, including members of the fibroblast growth factor (FGF) family. Both positive and negative effects of FGFs on dentinogenesis have been reported, but the underlying mechanisms of these conflicting results are still unclear. To gain a better insight into the role of FGF2 in dentinogenesis, we used dental pulp cells from various transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast differentiation. Our results showed that the continuous exposure of pulp cells to FGF2 inhibited mineralization and revealed both the stimulatory and inhibitory effects of FGF2 on the expression of markers of dentinogenesis and various transgenes. During the proliferation phase of in vitro growth, FGF2 increased the expression of markers of dentinogenesis and the percentages of dentin matrix protein 1/green fluorescent protein (DMP1-GFP)-positive functional odontoblasts and dentin sialophosphoprotein (DSPP)-Cerulean-positive odontoblasts. Additional exposure to FGF2 during the differentiation/mineralization phase of in vitro growth decreased the extent of mineralization and the expression of markers of dentinogenesis and of the DMP1-GFP and DSPP-Cerulean transgenes. Recovery experiments showed that the inhibitory effects of FGF2 on dentinogenesis were related to the blocking of the differentiation of cells into mature odontoblasts. These observations together showed the stage-specific effects of FGF2 on dentinogenesis by dental pulp cells, and they provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration.

Members of the fibroblast growth factor (FGF) family play essential and important roles in primary and reparative dentinogenesis, with conflicting results regarding their effects on odontoblast differentiation. Our recent studies showed that the effects of FGF2 on cells in odontoblast lineage were stage-specific and depended on the stage of cell maturity. Continuous exposure of pulp cells to FGF2 inhibited odontoblast differentiation, whereas early and limited exposure of pulp cells to FGF2 resulted in marked increases in odontoblast differentiation. The purpose of this study was to evaluate the cellular and molecular mechanisms regulating the inhibitory effects of FGF2 on odontoblast differentiation. To do so, we examined the effects of the addition of FGF2 during the differentiation/mineralization phase of the in vitro growth of pulp cultures derived from a series of green fluorescent protein reporter transgenic mice that display stage-specific activation of transgenes during odontoblast differentiation. Our results showed that this treatment first stimulated the differentiation of remaining progenitors in pulp cultures into functional odontoblasts but prevented their differentiation into mature odontoblasts. In addition, this treatment inhibited expression of markers of osteogenesis. Furthermore, we demonstrated that the inhibitory effects of FGF2 on odontoblast differentiation were mediated through activation of FGFR/MEK/Erk1/2 signaling and downregulation of bone morphogenetic protein signaling, with negative and positive roles in the expression of Dmp1 and Dspp, respectively, during the advanced stage of odontoblast differentiation.

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.

Aim To examine the feasibility of using the pOBCol3.6GFPtpz (3.6-GFP) transgenic mice as an in vivo model for studying the biological sequence of events during pulp healing and reparative dentinogenesis. Methodology Pulp exposures were created in the first maxillary molar of 12-16 week old 3.6-GFP transgenic mice with CD1 and C57/Bl6 genetic background. Direct pulp capping on exposed teeth were performed using mineral trioxide aggregate (MTA) followed by restoration with a light-cured adhesive system (AS) and composite resin. In control teeth, the AS was placed in direct contact with the pulp. Animals were euthanized at various time points after pulp exposure and capping. The maxillary arch was isolated, fixed and processed for histological and epifluorescence analysis to examine reparative dentinogenesis. Results Analysis of teeth immediately after pulp exposure revealed absence of odontoblasts expressing 3.6-GFP at the injury site. Evidence of reparative dentinogenesis was apparent at 4 weeks in 3.6-GFP mice in CD1 background and at 8 weeks in 3.6-GFP mice with C57/Bl6 background. The reparative dentine with both groups contained newly formed atubular-mineralized tissue resembling a dentine bridge and/or osteodentine that was lined by cells expressing 3.6-GFP as well as 3.6-GFP expressing cells embedded within the atubular matrix. Conclusion This study was conducted in a few animals and did not allow statistical analysis. The results revealed that the 3.6-GFP transgenic animals provide a unique model for direct analysis of cellular and molecular mechanisms of pulp repair and tertiary dentinogenesis in vivo. The study also shows the effects of the capping material and the genetic background of the mice in the sequence and timing of reparative dentinogenesis.

Dental caries (tooth decay) is the most common chronic disease. Dental tissue engineering is a promising alternative approach to alleviate the shortcomings of the currently available restorative materials. Mimicking the natural extracellular matrix (ECM) could enhance the performance of tissue engineering scaffolds. In this study, we developed microtubular (~20 μm diameter) polymethyl methacrylate (PMMA) scaffolds resembling the tubular (~2.5 μm diameter) structure of dentin, the collagen-based mineralized tissue that forms the major portion of teeth, to study the effect of scaffold architecture on differentiation of mouse dental pulp cells in vitro. Flat (control), plasma-treated solid and microtubular PMMA scaffolds with densities of 240±15, 459±51 and 480±116 tubules/mm2 were first characterized using scanning electron microscopy and contact angle measurements. Dental pulp cells were cultured on the surface of the scaffolds for up to 21 days and examined using various assays. Cell proliferation and mineralization were examined using Alamar Blue and Xylenol Orange (XO) staining assays, respectively. The differentiation of pulp cells into odontoblasts was examined by immunostaining for Nestin and by quantitative PCR analysis for dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and osteocalcin (Ocn). Our results showed that the highest tubular density scaffolds significantly (p<0.05) enhanced differentiation of pulp cells into odontoblasts as compared to control flat scaffolds, as evidenced by increased expression of Nestin (5.4x). However, mineralization was suppressed on all surfaces, possibly due to low cell density. These results suggest that the microtubular architecture may be a desirable feature of scaffolds developed for clinical applications. Lay Summary Regenerative engineering of diseased or traumatized tooth structure could avoid the deficiencies of traditional dental restorative (filling) materials. Cells in the dental pulp have the potential to differentiate to dentin-producing odontoblast cells. Furthermore, cell-supporting scaffolds that mimic a natural extracellular matrix (ECM) are known to influence behavior of progenitor cells. Accordingly, we hypothesized that a dentin-like microtubular scaffold would enhance differentiation of dental pulp cells. The hypothesis was proven true and differentiation to odontoblasts increased with increasing density of the microtubules. However, mineralization was suppressed, possibly due to a low density of cells. The results demonstrate the potential benefits of a microtubular scaffold design to promote odontoblast cells for regeneration of dentin.