Re-oxygenation improves hypoxia-induced pulp cell arrest.

Dental pulp cells can be exposed to hypoxia during severe inflammation or restorative procedures, though their response to hypoxia is not well-understood. We hypothesized that hypoxia has effects on the growth of pulp cells in vitro. When the cells were exposed to hypoxia for 48 hr, cell growth was suppressed, and cell death was detected by Hoechst staining. Western blot analysis revealed that phosphorylation of retinoblastoma protein was inhibited in cells exposed to hypoxia. Analyses of the molecules involved in retinoblastoma protein phosphorylation revealed that hypoxia suppressed cyclin D2 and activated p21(CIP1/WAF1). Further, hypoxia-exposed pulp cells showed improvement of cell viability, cell-cycle progression, and expression of cyclin D2 with re-oxygenation. These findings indicate that hypoxia-induced cell cycle arrest in pulp cells is reversible, while cyclin D2 may play an essential role in the improvement of cell proliferation with re-oxygenation.

Expressions of c-jun and jun-B proto-oncogenes in odontoblasts during development of bovine tooth germs.

c-jun and jun-B genes are among the nuclear proto-oncogenes induced by growth factors such as the TGF-beta superfamily and play important roles in cell differentiation. These gene products enhance expressions of proteins including osteocalcin, alkaline phosphatase, and collagens. On the other hand, it is well-known that the TGF-beta superfamily affects odontoblast differentiation, and that differentiated odontoblasts express extracellular and membrane proteins as described above. However, there are few reports of factors that participate in the transcriptional regulation of odontoblasts. Especially, little is known about the expression of c-jun and jun-B genes. In this study, we focused on the examination of expressions of c-jun and jun-B genes in dental papillae of bovine tooth germs. Using in situ hybridization, we found that these genes were expressed only in the odontoblastic lineage, but not in other dental papilla cells. Levels of c-jun and jun-B mRNAs increased along the gradient of differentiation of odontoblasts. These levels of c-jun mRNAs were maintained in both young and mature odontoblasts. However, unlike the c-jun gene, expression of the jun-B gene became sparse in mature odontoblasts compared with young odontoblasts. For further analysis, Northern hybridization of total RNA extracted from differentiated odontoblasts was performed for the examination of levels of jun-B mRNAs, indicating that levels of jun-B mRNAs of mature odontoblasts were clearly less than those of young odontoblasts. These results suggest that c-jun and jun-B genes may participate in the transcriptional regulation of odontoblasts of bovine tooth germs, and may control the odontoblast phenotype. Furthermore, our results suggest that these genes can be markers of odontoblasts during dentinogenesis; especially, high expression of jun-B gene can be a marker of young odontoblasts that start to form the new dentin matrix.

Presence of vesicles containing lactate dehydrogenase in the dentin of bovine tooth germs.

The dentin was removed from bovine tooth germs, followed by the separation of the extracellular matrix vesicle fraction after collagenase treatment. Lactate dehydrogenase (LDH)-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of odontoblasts. Other cytosolic enzymes were not detected in LDH-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles.

Temporal and spatial expression of c-jun and jun-B proto-oncogenes in pulp cells involved with reparative dentinogenesis after cavity preparation of rat molars.

c-jun and jun-B are nuclear proto-oncogenes induced by growth factors such as bone morphogenetic proteins (BMPs). These gene products enhance the expression of many genes, including osteocalcin and collagen types, indicating that c-jun and jun-B play important roles in the cell differentiation process. It is also known that BMPs affect the differentiation of pulp cells to odontoblast-like cells during reparative dentinogenesis, but little is known about the transcriptional regulation of genes in cells associated with reparative dentinogenesis. In this study, we examined the expression of c-jun and jun-B in pulp cells during reparative dentinogenesis after cavity preparation of rat molars by in situ hybridization. In rat tooth germs, c-jun and jun-B were co-expressed in the odontoblastic lineage. In rat adult molars, c-jun was expressed in the odontoblast layer, but the jun-B expression was absent in all pulp cells. After cavity preparation, we found that c-jun and jun-B were coexpressed in pulp cells underneath cavities. During the early phase of reparative dentinogenesis, levels of c-jun and jun-B greatly increased in pulp cells within and around the reparative dentin matrix formed adjacent to the cavity floor. Fourteen days after cavity preparation, c-jun and jun-B were expressed only in pulp cells lining the irregular surface of the thick reparative dentin. These results suggest that c-jun and jun-B may play important roles both in physiological and in reparative dentinogenesis; in particular, the limited distribution of the jun-B expression suggests a specific role of jun-B only in cells involved with the active formation of the dentin matrix during primary and reparative dentinogenesis.

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.

Primary and secondary induction of apoptosis in odontoblasts after cavity preparation of rat molars.

The death regulation of damaged pulp cells after cavity preparation is not well-known. In this study, we examined whether apoptosis is associated with the death regulation of damaged pulp cells. In normal rat molars, terminal deoxynucleotidyl transferase-mediated labeling (TUNEL)-positive cells were not observed. Just after surgery, odontoblasts under cavities were TUNEL-positive, and these signals disappeared in six hours. One day after surgery, we found the reappearance of TUNEL-positive cells in the subodontoblastic region under cavities, and positive signals disappeared in four days. Ultrastructure of TUNEL-positive cells showed characteristics typical of apoptotic cells. Phagocytosis of apoptotic cells by scavenger cells was also observed. By immunohistochemistry, we also found Bcl-2-positive odontoblasts one day after surgery. These results suggest that two waves of apoptosis are induced in odontoblasts after cavity preparation, and that apoptotic cells must be eliminated before the initiation of reparative dentinogenesis.

Regional measurement of resin-dentin bonding as an array.

During the development of the microtensile bond-testing method, large variations in bond strengths were noted among serial sections. The reason for these variations is unknown. The purpose of this work was to determine the consistency of resin-dentin bond strengths across the occlusal surface of coronal dentin by dividing composite resin buildups into an array of 1x1 mm beams, the top half consisting of composite resin, and the bottom half consisting of dentin. Extracted human third molars had the occlusal enamel removed as a single section by means of a diamond saw. Resin composite buildups were made after the dentin was bonded with either One-Step or MacBond. After being stored in 37 degrees C water for 1 day, the teeth were vertically sectioned at 1-mm increments into slabs of bonded teeth. Each slab was further subdivided by vertical sections into 1x1x8 mm beams. Each beam was assigned an x-y coordinate and tested for tensile bond strength. Two different clinicians (A and B) performed the same procedures using One-Step in a parallel study. Using One-Step, clinician A obtained a large number of zero bonds in superficial dentin but fewer in deep dentin. This resulted in a very large standard deviation in bond strengths (mean +/- SD of 22+/-20 MPa in superficial dentin and 27+/-14 MPa in deep dentin). Clinician B obtained much higher (p<0.001) and more uniform bond strengths with One-Step (56+/-13 MPa in superficial dentin and 57+/-12 MPa in deep dentin). With MacBond, there were no zero bonds and hence less variation, with a mean of 41+/-13 MPa in superficial dentin and 27+/-12 MPa (x +/- SD) in deep dentin. When pairs of Z100 resin composite cylinders were bonded together with One-Step and then sectioned into an array, there was little variation in regional bond strength (37 +/-1 MPa). Dividing bonded resin composite buildups into an array of 20 to 30 1x1x8 mm beams allows for the evaluation of uniformity of resin-dentin bonds. The method used in this study detected local regional differences in resin-dentin bond strengths. The largest differences were shown to be related to technique rather than to material. The results indicate that resin-dentin bonds may not be as homogenous as was previously thought.

The presence of vesicles containing lactate dehydrogenase in the ameloblast layer of bovine enamel organ.

Ameloblast layers were removed from bovine enamel organs, followed by the separation of the extracellular matrix-vesicle fraction after collagenase digestion. Lactate dehydrogenase (LDH)-containing vesicles were found in that fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of ameloblasts, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles. The existence of LDH-containing vesicles in enamel organ, where mineralization had been believed not to be initiated by matrix vesicles, suggests the possibility that LDH-containing vesicles have a specific function different from that of matrix vesicles.

Effects of cross-sectional area on resin-enamel tensile bond strength.

OBJECTIVE: It was hypothesized that there is an inverse relationship between resin-enamel bond strength and bonded cross-sectional area, and that there are regional differences in resin-enamel bond strength. METHODS: The facial and lingual surfaces of extracted human third molars were ground down 0.3 mm using 240 grit abrasive paper and were then bonded with either Clearfil Liner Bond 2 or Scotchbond Multi-Purpose Plus adhesive systems using the manufacturer's instructions. The bonded surfaces then received a resin composite build-up. After 24 h of storage in water, the bonded teeth were vertically serially sectioned into 1.0 mm thick slabs using a diamond saw, and the bonded surface area at the resin-enamel interface was varied from 0.5 to 3.0 mm2 using a diamond saw under microscopic observation. The trimmed region was varied from the occlusal third of the facial or lingual enamel to the middle third, to the cervical third. The trimmed specimens were then glued to a Bencor Multi-T device, placed in an Instron testing machine and stressed to failure at 1 mm/min. A three-factor ANOVA was used to compare bond strengths (buccal vs. lingual, occlusal vs. middle vs. cervical-third, vs. materials). Regression analysis was used to examine the relationship between bond strength and bonded cross-sectional area for each material on occlusal enamel. RESULTS: For both bonding systems, there was a highly significant (p < 0.001) inverse exponential relationship between tensile bond strength (y axis) and bonded cross-sectional area (x axis) with y intercepts of 51 and 59 MPa for Clearfill Liner Bond 2 and Multi-Purpose Plus, respectively. Using both materials, the highest bond strengths were measured in the occlusal third, which were significantly higher (p < 0.05) than those made to cervical enamel. SIGNIFICANCE: Like resin-dentin bonds, resin-enamel bonds exhibit an inverse relationship with cross-sectional area. This relationship becomes more apparent at bonded surface areas below 2 mm2 and is probably due to reductions in the number of interfacial stress-raisers as samples are made smaller.

Durability of resin-dentin bonds.

PURPOSE: The purpose of this study was to determine if the durability of resin-dentin bonds could be evaluated more quickly if the bond specimen was divided into 1 x 1 x 8 mm beams incubated at 37 degrees C for a 90-day period. MATERIALS AND METHODS: Extracted human third molars were prepared for bonding by removing the occlusal surface near the dento-enamel junction (superficial dentin group) or near the pulp (deep dentin group). The teeth were bonded either with MacBond, One Step or Clearfil Liner Bond 2, and then builtup to form a flat resin composite crown. After 24 hours in water, each buildup was vertically divided into slabs 1 mm thick, the top half of which was resin, with the bottom half as dentin. Each slab was then vertically sectioned at 1-mm increments to create 1 x 1 x 8-mm beams of resin-bonded dentin. They were incubated for 1 day or 90 days at 37 degrees C, followed by measurement of the tensile bond strengths. The results were analyzed by the Least-Squares Means method at the 95% confidence level. RESULTS: MacBond gave the highest (p < 0.05) 1-day bond strengths to superficial dentin, but significantly lower bond strengths were measured in deep dentin. There were no significant differences in the bond strengths of either One Step or Clearfil Liner Bond 2 to superficial vs deep dentin at 1 day, but at 90 days their bond strengths to deep dentin had fallen significantly (p < 0.05). Prepolymerized cylinders of resin composite bonded together with One Step showed little variation in bond strength over the 90-day experiment. SEM examination of the failed bonds showed increased porosity in intertubular dentin over time. CONCLUSION: The results indicate that division of large specimens into many small beams accelerated the deterioration of bond strength in deep dentin in all three bonding systems and in both superficial and deep dentin in the MacBond treated specimens. This method seems promising for studying the durability of resin-dentin bonds.

Fracture aspects of resin-dentin bonding in non-trimming microtensile test.

Comparative studies on resin-dentin bond strength and failure mode were performed between the conventional tensile test and the microtensile test with non-trimming small specimens, 1 x 1 mm in cross-section, for two brands of dentin bonding systems. The fracture surface of the conventional large specimen showed a catastrophic cohesive failure in dentin at its center and a lesser adhesive failure, suggesting that the whole failure was due to the development of some major cracks. The non-trimming microtensile test showed significantly larger average bond strength with markedly larger standard deviation and significantly larger fraction of adhesive failure than the conventional test. Some small specimens were extremely strong and some were weak according to the heterogeneous distribution of tight bonding and defective or deficient bonding over the whole dentin surface. These results suggest that the non-trimming microtensile test may potentially provide more realistic aspects of resin-dentin bonding than the conventional bulk specimen.

Evaluation of cytotoxicity of calcium phosphate cement consisting of alpha-tricalcium phosphate and dicalcium phosphate dihydrate.

A newly developed calcium phosphate cement, MM, was evaluated for tissue irritability by means of cell cultures. In this study, MM showed extensive mild cytotoxicity compared with other cements before setting. Inhibition of adhesion of L-929 cells was not observed after contact with MM for 24 hours. The influence of MM on colony formation was approximately the same as that of another calcium phosphate cement and less than that of a glass ionomer cement. Toxicity of MM after setting was compared with four cements; another calcium phosphate cement, glass ionomer cement, silicate cement and zinc oxide eugenol cement, but MM showed the least influence on cell morphology. Judging from these results, MM appears to be less cytotoxic than the cements in current use.

Modulation of the in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. III. Enhancement by RP-3 treatment of the anti-sheep red blood cell plaque-forming cell response in rats.

Because recent work has shown that neutrophils produce various cytokines and are activated by these agents, this study was undertaken to examine neutrophil participation in immune networks. In our previous work, we demonstrated that delayed type hypersensitivity to SRBC and accompanying mononuclear leukocyte recruitment are inhibited in vivo by selective depletion of neutrophils using a mAb designated RP-3. To elucidate the function of these cells in Ab production, we assessed the splenic plaque-forming cell response to SRBC in a rat model through selective depletion of circulating neutrophils by RP-3. This depletion which occurred at the time of immunization resulted in an increase in the number of anti-SRBC Ab producing cells, as determined by the direct and indirect plaque-forming cell response. This phenomenon was observed only when the Ag was administered i.p. and not with i.v. immunization. These results suggest that neutrophils suppress Ab production in certain situations.

Modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. II. Inhibition by RP-3 treatment of mononuclear leukocyte recruitment in delayed-type hypersensitivity to sheep red blood cells in rats.

We demonstrated in our previous paper that treatment of rats with RP-3, a mAb that depletes in vivo neutrophils selectively, resulted in inhibition of both the priming and effector phases of delayed type hypersensitivity (DTH) to SRBC. In order to clarify the mechanisms of this phenomenon, we examined the effect of RP-3 treatment on mononuclear leukocyte (MNL) recruitment in DTH. When rats had been treated with RP-3 at the time of Ag priming, MNL migration, which accompanies DTH, was inhibited. The prior infiltration of neutrophils was also partially required for MNL recruitment because migration was inhibited when the immune rats were treated with RP-3 at the time of DTH elicitation.

Disorder of salivary secretion in inbred polydipsic mouse.

To find mechanisms of an extreme polydipsia in an inbred strain of mice, STR/N, this study was undertaken using Institute of Cancer Research (ICR) mice as a control. During food deprivation, daily water intake of both strains decreased. The decrement in the STR/N mice was larger than that in the ICR mice. During dehydration, daily food intake of the STR/N mice was smaller than that of the ICR mice. These data indicate that prandial drinking was more severely affected for the STR/N mice. Under anesthesia, the stimulated salivary secretion by pilocarpine of the STR/N mice was significantly smaller than that of the ICR mice. The submandibular gland of the STR/N mice was lighter and harder than that of the ICR mice. After desalivation from the major three salivary glands, the ICR mice drank as much as the STR/N mice. Young STR/N mice with undeveloped polydipsia did not show different salivary secretion stimulated by pilocarpine from the young ICR mice. These findings indicate a dysfunction with age in the salivary glands of the STR/N mice, and they suggest that the decreased saliva induces thirst and triggers extraordinary drinking in the polydipsic mice.

Efficacy of ozone on survival and permeability of oral microorganisms.

In the present study, we examined the effect of ozonated water on oral microorganisms and dental plaque. Almost no microorganisms were detected after being treated with ozonated water (4 mg/l) for 10 s. To estimate the ozonated water-treated Streptococcus mutans, bacterial cells were stained with LIVE/DEAD BacLight Bacterial Viability Kit. Fluorescence microscopic analysis revealed that S. mutans cells were killed instantaneously in ozonated water. Some breakage of ozonated water-treated S. mutans was found by electron microscopy. When the experimental dental plaque was exposed to ozonated water, the number of viable S. mutans remarkably decreased. Ozonated water strongly inhibited the accumulation of experimental dental plaque in vitro. After the dental plaque samples from human subjects were exposed to ozonated water in vitro, almost no viable bacterial cells were detected. These results suggest that ozonated water should be useful in reducing the infections caused by oral microorganisms in dental plaque.

[Changes of ciscoelastic behaviour during curing of polyether rubber impression material].

To obtain a precise impression, it is indispensable to understand changes of physical property of the material used, during curing. The authors measured the viscosity and the storage modulus of polyether rubber which was recently introduced as a commercial impression material and tried to make clear the curing mechanism of the material. The less the amount of catalyst added to the base material, the more approximative is the behaviour to the first-order reaction. But according as the amount of catalyst increases the behaviour becomes not to be regarded as the first-order reaction, i.e., it is surmised that the curing reaction becomes so complicated. The more the amount of catalyst and the higher the temperature after mixing, the shorter is the setting time. The curing reaction of polyether rubber impression material is supposed to be completed in about 3 hours after mixing. Polyether rubber impression material showed the highest storage modulus among the three rubber impression materials not used, namely, polyether, polysulfide and silicone rubber. The values of this highest modulus of polyether rubber were about 2.4approximately2.8 times of those of polysulfide and silicone rubber impression material in 3 hours after mixing.

Enhancement of delayed-type hypersensitivity to sheep red blood cells in mice by granulocyte colony-stimulating factor administration at the elicitation phase.

We previously demonstrated that selective depletion of polymorphonuclear neutrophils by a mAb inhibits both the priming and effector phases of delayed-type hypersensitivity (DTH) to SRBC. To better understand the role of polymorphonuclear neutrophils in DTH, we examined the effect of granulocyte CSF (G-CSF) on DTH to SRBC in mice. G-CSF administered at the elicitation phase enhanced a DTH response that was weak in control G-CSF-untreated mice. This treatment also augmented mononuclear leukocyte recruitment in DTH in these mice. However, G-CSF administration failed to augment priming of DTH to SRBC under any conditions examined.

Modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. I. Inhibition by RP-3 treatment of the priming and effector phases of delayed type hypersensitivity to sheep red blood cells in rats.

Recent studies on neutrophils have revealed that these cells produce various cytokines, and may be involved in regulation of the immune response. We examined whether neutrophils are involved in delayed type hypersensitivity (DTH) to SRBC in rats by selective depletion of in vivo neutrophils using a mAb designated RP-3. When the rats had been treated with RP-3 at the time of priming with SRBC, DTH to these cells was inhibited. Furthermore, RP-3 treatment was effective in inhibiting the effector phase of the DTH response to SRBC. When spleen cells from rats that had been treated with RP-3 at the time of immunization were used for local transfer of DTH, footpad swelling was significantly less than that induced by spleen cells from the RP-3-untreated immune rats.