Takahiko Morotomi

Formation of Dentinal Bridge on Surface of Regenerated Dental Pulp in Dentin Defects by Controlled Release of Fibroblast Growth Factor–2 From Gelatin Hydrogels

INTRODUCTION Pulp regeneration therapy is important to overcome the limitations of conventional therapy to induce reparative dentinogenesis. In the present study, we examined the effects of controlled release of different dosages of fibroblast growth factor-2 (FGF-2) from gelatin hydrogels to regenerate the dentin-pulp complex. METHODS After the amputation of dental pulp of rat molars, gelatin hydrogels incorporating various dosages of FGF-2 were individually implanted into dentin defects above the sites of the amputated pulps. Histologic changes as well as the expression of dentin matrix protein-1 (DMP-1) and nestin in the dentin defect area above the amputated pulp were analyzed. RESULTS We found that controlled release of high doses of FGF-2 from gelatin hydrogels induced DMP-1-positive calcified particles in the proliferating pulp, whereas a moderate dose of FGF-2 induced DMP-1-positive dentinal bridge on the surface of the proliferating pulp. These findings indicate that the dosage of released FGF-2 has an influence on the structure of calcified tissue regenerated in dentin defects. In addition, pulp cells near calcified tissues regenerated in dentin defects were nestin-negative, suggesting that the calcified tissues might be osteodentin. CONCLUSIONS Our results showed that the dentin regeneration on amputated pulp, not reparative dentin formation toward amputated pulp, can be regulated by adjusting the dosage of FGF-2 incorporated in biodegradable gelatin hydrogels.

Characterization of Dental Epithelial Progenitor Cells Derived from Cervical-loop Epithelium in a Rat Lower Incisor

Dental epithelial progenitor cells differentiate into various cell types during development of tooth germs. To study this mechanism, we produced immortalized dental epithelial progenitor cells derived from the cervical-loop epithelium of a rat lower incisor. The expression patterns of cytokeratin 14, nerve growth factor receptor p75, amelogenin, Notch2, and alkaline phosphatase were examined by immnohistochemistry in both lower and higher cell densities. The patterns of each were compared in the dental epithelium of rat lower incisors. The results demonstrated that these cells could produce ameloblast lineage cells, stratum intermedium cells, stellate reticulum, and outer enamel epithelium. Furthermore, fibroblast growth factor 10 stimulated proliferation of dental progenitor cells and subsequently increased the number of cells expressing alkaline phosphatase. These results suggest that fibroblast growth factor 10 plays a role in coupling mitogenesis of the cervical-loop cells and the production of stratum intermedium cells in rat incisors.

Establishment of Dental Epithelial Cell Line (HAT-7) and the Cell Differentiation Dependent on Notch Signaling Pathway

Rat incisors grow continuously throughout life. Producing a variety of dental epithelial cells is performed by stem cells located in the cervical loop of the incisor apex. To study the mechanisms for cell differentiation, we established a dental epithelial cell line (HAT-7) originating from a cervical loop epithelium of a rat incisor. Immunochemical studies showed that HAT-7 produced the cells expressing amelogenin, ameloblastin, or alkaline phosphatase (ALP). To illustrate a role of Notch signaling in the determinant of the cell fate, we examined expression patterns of Notch1 and Jagged1 in HAT-7 density dependently. At lower cell density, Notch1- or Jagged1-expressing cells were not seen. However, when they were fully confluent, cells began to express Notch1 or Jagged1 strongly. Some ALP-positive cells were almost consistent with Notch1-expressing cells but not Jagged1-expressing cells. These results suggested that the determinant of direction of differentiation was associated with Notch signaling pathway.

Effects of hyaluronic acid sponge as a scaffold on odontoblastic cell line and amputated dental pulp.

It is important to develop a suitable three-dimensional scaffold for the regeneration therapy of dental pulp. In the present study, the effects of hyaluronic acid (HA) sponge on responses of the odontoblastic cell line (KN-3 cells) in vitro, as well as responses of amputated dental pulp of rat molar in vivo, were examined. In vitro, KN-3 cells adhered to the stable structure of HA sponge and that of collagen sponge. In vivo, dental pulp proliferation and vessel invasion were observed in both sponges implanted at dentin defect area above amputated dental pulp, and the cell-rich reorganizing tissue was observed in the dentin defect when HA sponge was implanted as compared with collagen sponge. Expression levels of IL-6 and TNF-alpha in KN-3 cells seeded in HA sponge were nearly the same with those in the cells seeded in collagen sponge, while the numbers (0.67 x 10(3) at 1 week and 0.7 x 10(3) at 3 weeks) of granulated leukocytes that invaded into HA sponge from amputated dental pulp was significantly lower than those (1.22 x 10(3) at 1 week and 1.1 x 10(3) at 3 weeks) of collagen sponge (p < 0.01 at 1 week and p < 0.05 at 3 weeks). These results suggest that HA sponge has an appropriate structure, biocompatibility, and biodegradation for use as a scaffold for dental pulp regeneration.

Transient Co-localization of c-Jun N-terminal Kinase and c-Jun with Heat Shock Protein 70 in Pulp Cells during Apoptosis

The c-Jun N-terminal kinase (JNK) pathway and heat-shock proteins (HSPs) are involved in stress-induced apoptosis. Here we examined the association of JNK, c-Jun, and anti-apoptotic HSPs with pulp apoptosis during wound healing. In normal pulp, c-Jun was activated only in a few pulp cells, but JNK was not. HSP70 was expressed in the cytoplasm of pulp cells. One day after injury, active JNK and c-Jun were detected in apoptotic pulp cells, whereas HSP70 was detected in non-apoptotic cells. We also found the translocation of HSP70 into nuclei of pulp cells, and co-localization with active JNK and c-Jun. Four days after injury, active JNK and c-Jun disappeared in pulp cells, and HSP70 was relocalized from nuclei to the cytoplasm. These results suggest that the JNK pathway may be one of the compartments inducing apoptosis in pulp cells, and that HSP70 may have an inhibitory role in the apoptosis of pulp cells during wound healing.

Effects of sequential exposure to lipopolysaccharide and heat stress on dental pulp cells

In the present study, we examined the effects of sequential exposure to bacterial lipopolysaccharide (LPS) and heat stress on dental pulp cells. LPS induced the proliferation of pulp cells through the activation of p38 MAPK. HSP27 was expressed in cells with or without LPS during the entire period of heat stress, while transiently phosphorylated by short‐term heat stress. In LPS‐treated cells, short‐term heat stress also induced the phosphorylation of HSF1. The immediate phosphorylation of HSF1 and HSP27 in LPS‐treated cells by short‐term heat stress occurred dependent on the activation of p38 MAPK. However, with long‐term heat stress, the activation of HSF1 and induction of HSP27 occurred independent of p38 MAPK. Further, full activation of Akt in LPS‐treated cells was immediately induced by short‐term heat stress and lasted during the entire period of heat stress. IκBα was induced and phosphorylated throughout sequential exposure to LPS and heat stress. These results suggest that LPS has the unique effects on the cytoprotection and the cell death of pulp cells during heat stress through the modification and the activation of heat stress responsive molecules, HSF1 and HSP27, and cell survival molecules, Akt and NF‐κB/IκBα. J. Cell. Biochem. 99: 797–806, 2006.

Differential Induction of Apoptosis by Capping Agents during Pulp Wound Healing

Effects of capping agents on two waves of apoptosis during pulp wound healing were examined. After cavity preparation of rat molars, cavities were filled with calcium hydroxide, zing oxide eugenol cement, or 4-META/MMA-TBB resin (4MMT) and some were unfilled. One hour or 1 day after filling, we examined the distribution and the number of apoptotic cells by terminal deoxynucleotidyl transferase-mediated labeling (TUNEL) assay. One hour after filling, there were no differences in distribution patterns or the number of apoptotic odontoblasts among the four groups. One day after filling, we found differences in distribution patterns and the number of apoptotic pulp cells among the four groups. Especially in the 4MMT group, the distribution pattern of apoptotic cells was more broadly spread, and the number of apoptotic cells was significantly larger than those of other groups. These results suggest that capping agents may have an effect on pulp apoptosis and that 4MMT may actively induce apoptosis during pulp wound healing.

Possible Involvement of Smad Signaling Pathways in Induction of Odontoblastic Properties in KN-3 Cells by Bone Morphogenetic Protein-2: A Growth Factor to Induce Dentin Regeneration

We examined the effects of bone morphogenetic protein-2 (BMP-2) on growth, differentiation, and intracellular signaling pathways of odontoblast-like cells, KN-3 cells, to clarify molecular mechanisms of odontoblast differentiation during pulp regeneration process. After treatment with BMP-2, the cell morphology, growth, alkaline phosphatase (ALP) activity, and the activation and expression of BMP-induced intracellular signaling molecules, such as Smad1/5/8 and Smad6/7, as well as activities of dentin sialoprotein (DSP) and dentin matrix protein 1 (DMP1), were examined. BMP-2 had no effects on the morphology, growth, or ALP activity of KN-3 cells, whereas it induced the phosphorylation of Smad1/5/8 and expression of Smad6/7. BMP-2 also induced the expressions of DSP and DMP-1. Our results suggest that KN-3 cells may express an odontoblastic phenotype with the addition of BMP-2 through the activation of Smad signaling pathways.

Effects of heat stress and starvation on clonal odontoblast-like cells.

INTRODUCTION Heat stress during restorative procedures, particularly under severe starvation conditions, can trigger damage to dental pulp. In the present study, we examined effects of heat stress on odontoblastic activity and inflammatory responses in an odontoblast-like cell line (KN-3) under serum-starved conditions. METHODS Viability, nuclear structures, and inflammatory responses of KN-3 cells were examined in culture medium containing 10% or 1% serum after exposure to heat stress at 43°C for 45 minutes. Gene expression of extracellular matrices, alkaline phosphatase activity, and detection of extracellular calcium deposition in cells exposed to heat stress were also examined. RESULTS Reduced viability and apoptosis were transiently induced in KN-3 cells during the initial phases after heat stress; thereafter, cells recovered their viability. The cytotoxic effects of heat stress were enhanced under serum-starved conditions. Heat stress also strongly up-regulated expression of heat shock protein 25 as well as transient expression of tumor necrosis factor-alpha, interleukin-6, and cyclooxygenase-2 in KN-3 cells. In contrast, expression of type-1 collagen, runt-related transcription factor 2, and dentin sialophosphoprotein were not inhibited by heat stress although starvation suppressed ALP activity and delayed progression of calcification. CONCLUSIONS Odontoblast-like cells showed thermoresistance with transient inflammatory responses and without loss of calcification activity, and their thermoresistance and calcification activity were influenced by nutritional status.

Current and future options for dental pulp therapy

Summary Dental pulp is a connective tissue and has functions that include initiative, formative, protective, nutritive, and reparative activities. However, it has relatively low compliance, because it is enclosed in hard tissue. Its low compliance against damage, such as dental caries, results in the frequent removal of dental pulp during endodontic therapy. Loss of dental pulp frequently leads to fragility of the tooth, and eventually, a deterioration in the patient’s quality of life. With the development of biomaterials such as bioceramics and advances in pulp biology such as the identification of dental pulp stem cells, novel ideas for the preservation of dental pulp, the regenerative therapy of dental pulp, and new biomaterials for direct pulp capping have now been proposed. Therapies for dental pulp are classified into three categories; direct pulp capping, vital pulp amputation, and treatment for non-vital teeth. In this review, we discuss current and future treatment options in these therapies.