Journal of Dental Research

Prion diseases have recently emerged as a significant challenge to health-care workers, including those involved in dentistry. Abnormal prion proteins are resistant to complete inactivation by conventional sterilization techniques. In the last decade, a new form of prion disease emerged in the UK, termed "variant CJD", thought to be acquired by consumption of bovine spongiform encephalopathy-contaminated food products. At present, CJD is an invariably fatal disease with no immediate prospect of treatment or vaccination. Of concern with the variant form of CJD, unlike the more classic forms of the disease, is the appearance of significant levels of infectivity outside the central nervous system. This raises concerns for the potential transmission of prion proteins via surgical procedures from individuals in the asymptomatic stage of the disease. This article reviews the existing knowledge base on the nature of prions, their distribution in oral tissues, and the implications for dental treatment.

Dentin Matrix Protein 1 (Dmp1) was originally identified from dentin. However, its expression and function in vivo are not clear. To clarify these two issues, we have generated mice carrying a truncated Dmp1 gene by using gene targeting to replace exon 6 with a lacZ gene. Northern blot analysis shows the expected 5.8-kb Dmp1-lacZ fusion transcript and loss of the wild-type 2.8-kb Dmp1 transcript, confirmed by a lack of immunostaining for the protein. Using heterozygous animals, we demonstrate that Dmp1 is specific for mineralized tissues. Not previously shown, Dmp1 is also expressed in pulp cells. Dmp1-deficient embryos and newborns display no apparent gross abnormal phenotype, although there are a modest expansion of the hypertrophic chondrocyte zone and a modest increase in the long bone diameter. This suggests that DMP1 is not essential for early mouse skeletal or dental development.

The structural similarity of eugenol with capsaicin suggests that these two agents may share molecular mechanisms to produce their effects. We investigated the effects of eugenol in comparison with those of capsaicin using whole-cell patch clamp and Fura-2-based calcium-imaging techniques in a heterologous expression system and with sensory neurons. In vanilloid receptor 1 (VR1)-expressing human embryonic kidney (HEK) 293 cells and trigeminal ganglion (TG) neurons, eugenol activated inward currents, whereas capsazepine, a competitive VR antagonist, and ruthenium red (RR), a functional VR antagonist, completely blocked eugenol-induced inward currents. Moreover, eugenol caused elevation of [Ca²⁺]_i, and this was completely abolished by both capsazepine and ruthenium red in VR1-expressing HEK 293 cells and TG neurons. Our results provide strong evidence that eugenol produces its effects, at least in part, via VR1 expressed by the sensory nerve endings in the teeth.

We noted the absence of all 4 third molars (M3) in Epilepsy-Like disorder (EL) mice, an animal model for the study of epilepsy. This study was conducted to identify the major candidate chromosome and to detect the region that included the candidate gene causing the absence of M3 in EL mice. Linkage analysis was performed on genetic crosses of EL mice and MSM (Mus musculus molossinus) strain mice, which had a normal complement of teeth. Genome-wide screening by individual genotyping of F₂ intercross mice identified mouse chromosome 3 as one of the candidate chromosomes. Based on high linkage scores in detailed genotyping of F₂ intercross and N₂ backcross mice, the candidate locus for the absence of M3 in EL mice was mapped on the middle of chromosome 3.

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In addition to the well-established catabolic effects (activation of bone resorption) of PTH, it is now recognized that intermittent PTH administration has anabolic effects (promotion of bone formation). The aim of this study was to investigate whether intermittent administration of PTH in rodents would block the alveolar bone loss observed in rats when a ligature model of periodontitis is used. Morphometric analysis showed that intermittent PTH administration (40 μg/kg) was able to protect the tooth site from periodontitis-induced bone resorption. In addition, there was a significant reduction in the number of inflammatory cells at the marginal gingival area in sections obtained from animals receiving PTH compared with control animals. These findings demonstrated that intermittent PTH administration was able to protect against periodontitis-associated bone loss in a rodent model.

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.

15-deoxy-Δ^12,14-prostaglandin J₂ (15-d-PGJ₂) and troglitazone have been shown to induce apoptosis in several carcinoma cell lines. However, apoptotic signaling pathways of these agents are poorly understood. We tested the hypothesis that peroxisome proliferator-activated receptor-γ ligands such as these two agents will induce caspase-mediated apoptosis in human oral squamous cell carcinomas (SCC). Treatment of these cell lines with 15-d-PGJ₂ or troglitazone decreased cell viability in a time- and dose-dependent manner. 15-d-PGJ₂, but not troglitazone, induced apoptosis, and this effect was time-dependent. Exposure of cells to 20 μM of 15-d-PGJ₂ initiated early cytochrome c release, followed by late caspase activation. Furthermore, co-treatment with caspase inhibitors such as Z-VAD-FMK or Z-DEVD-FMK of oral SCC cells that had been treated with 20 μM of 15-d-PGJ₂ blocked apoptosis. Our study demonstrates that treatment with 15-d-PGJ₂, but not troglitazone, induces apoptosis in human SCC cell lines, and 15-d-PGJ₂ appears to work through cytochrome c release and caspase activation.

Many different factors can lead to inflammatory changes within temporomandibular joint tissues. This investigation examined if the expression of TNF-α and its receptors was altered in TMJ tissues during inflammation. Adult male rats were injected bilaterally with complete Freund’s adjuvant (CFA) into the TMJ or served as uninjected controls and were killed two days after CFA treatment. TMJ tissues were removed, and expression of TNF-α and its receptors was examined via gene microarray analysis, RT-PCR, Western blot, and ELISA. Gene microarray analysis provided evidence for changes in gene expression, notably that TNF-α and TNF-R1, but not TNF-R2, were significantly elevated in CFA-treated TMJ tissues. However, protein levels of TNF-α, TNF-R1, and TNF-R2 were all significantly increased in CFA-treated TMJ tissues. These results indicate that the pro-inflammatory cytokine TNF-α may play a significant role in the onset of inflammatory conditions associated with adjuvant-induced arthritis of the TMJ.

Previous in vivo studies have revealed that resins may generate a persistent inflammation of oral tissues and cell death as well. Apoptosis is an important regulated process that results in rapid cell death. This study tested the hypothesis that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) causes apoptosis. The effects of TEGDMA on proliferation and apoptosis in primary oral fibroblasts were analyzed by light microscopy and flow cytometry (FACS; Annexin V-assay). TEGDMA at 5 and 7.5 mM inhibited proliferation after 24 hrs. No increased frequency of apoptosis or necrosis was observed with 1 mM or 2.5 mM TEGDMA after 24 hrs. Apoptosis and Annexin V-positive cells were observed with 5 mM and 7.5 mM TEGDMA by light microscopy after 24 hrs. A dramatic increase in apoptotic cells was detected by FACS after 24 hrs with 7.5 mM TEGDMA. Thus, TEGDMA was cytotoxic and “apoptotic” in a dose- and time-dependent manner.

The mechanical properties of cancellous bone depend on the bone structure. The present study examined the extent to which the apparent stiffness of the cancellous bone of the human mandibular condyle can be predicted from its structure. Two models were compared. The first, a structure model, used structural parameters such as bone volume fraction and anisotropy to estimate the apparent stiffness. The second was a finite element model (FEM) of the cancellous bone. The bone structure was characterized by micro-computed tomography. The calculated stiffnesses of 24 bone samples were compared with measured stiffnesses. Both models could predict 89% of the variation in the measured stiffnesses. From the stiffness approximated by FEM in combination with the measured stiffness, the stiffness of the bone tissue was estimated to be 11.1 ± 3.2 GPa. It was concluded that both models could predict the stiffness of cancellous bone with adequate accuracy.

In this study, we explored the reduction of shrinkage stresses in restored teeth by stimulating viscous flow of adhesive restoratives during curing, by increasing the TEGDMA/BisGMA ratio in the resin of composite restoratives. We studied a series of experimental two-paste composites with different amounts of TEGDMA (30, 50, 70 wt%, respectively) in the resin by mechanical testing, infrared spectroscopy, and dilatometry, to determine how comonomer composition affects the mechanical and chemical properties of the composite during curing. It was found that the polymerization rate of BisGMA-TEGDMA composites is indicative of the viscoelastic behavior during curing: The higher the reactivity, the higher the stiffness and viscosity development. Composites with 50 wt% TEGDMA in the resin displayed the highest maximum polymerization rate. High amounts of TEGDMA in the resin only modestly increased the pre-gel viscous flow (= lowered viscosity) property of composites. Of these composites, high post-gel shrinkage is the decisive factor in high shrinkage stress development.

Fluoride is added to many dental restorative materials, including glass-ionomer cements, for the specific purpose of leaching fluoride into the surrounding tissues to provide secondary caries inhibition. During the caries process, an acidic environment attacks the dental tissues as well as the glass-ionomer cement. We hypothesized that pH significantly affects the rate of release of fluoride from the glass-ionomer cement. A continuous-flow fluoride-measuring system that monitors the amount of fluoride released over time was used to determine the release of fluoride from a resin-modified glass-ionomer cement (KetacFil^®). The results show that the release rate began with a fast burst of fluoride which quickly diminished to low levels in 3 days. Under neutral pH conditions, the rate of fluoride release at 72 hrs was significantly slower than at pH 4.

Extensive research suggested that calcium phosphate (CaP) coatings on titanium implants are essential for early bone response. However, the characterization of CaP crystallinity and the means to control coating crystallinity are not well-established. In this study, the effect of a 400°C heat treatment for 1, 2, or 4 hours, and in the presence or absence of water vapor, on CaP crystallinity was investigated. Scanning electron microscopy indicated dense as-sputtered coatings. Increase in coating crystallinity was observed to be consistent with the increasing number of PO₄ peaks observed as a result of different heat treatments. In addition, x-ray diffraction analyses indicated amorphous as-sputtered coatings, whereas crystalline CaP coatings in the range of 0-85% were observed after different post-deposition heat treatments. It was concluded that the presence of water vapor and post-deposition heat treatment time significantly affect the crystallinity of CaP coatings, which may ultimately affect bone healing.

Low bone mineral density and rapid bone loss of the skeleton are associated with mortality risk from vascular diseases in post-menopausal women. Panoramic radiographic measurements are considered as indicators of skeletal bone mineral density or bone turnover. We hypothesize that such measurements may be associated with vascular disease risk in post-menopausal women. Associations of mandibular cortical shape and width on panoramic radiographs with skeletal bone mineral density and risk factors related to vascular diseases were investigated in 87 post-menopausal women. Cortical shape was associated with skeletal bone mineral density, low-density lipoprotein cholesterol, apolipoprotein B, resting heart rate, and endothelial dysfunction. Cortical width was associated with skeletal bone mineral density, low-density lipoprotein cholesterol, and apolipoprotein A1. Dentists may be able to refer women with increased risk of vascular diseases, as well as low bone mineral density, to medical professionals for further examination by panoramic findings.

A loss of acinar cells occurs with aging, while salivary production remains age-stable in healthy adults. It is hypothesized that a secretory reserve exists to preserve function despite a loss of acinar cells in normal aging. The purpose of this double-blind, placebo-controlled, crossover study was to determine age-related differences in salivary response to an anti-sialogogue (glycopyrrolate). Thirty-six healthy subjects (18 young - 20-38 yrs; 18 older - 60-77 yrs) received 4.0 μg/kg IV glycopyrrolate. Parotid and submandibular/sublingual saliva samples and xerostomia questionnaire responses were collected. Variables calculated for each subject were: times to initial and maximum suppression and xerostomic complaint; time to recovery; and durations of suppression and complaint. Salivary function was more adversely affected in older persons. There were no consistent age-associated questionnaire response differences. These findings suggest that salivary gland output is more adversely affected by an anti-sialogogue in healthy older vs. younger adults, supporting the secretory reserve hypothesis of salivary function.

Dioxin, a powerful hormone-disrupting chemical, exhibits serious health effects when it reaches body fat. Here we analyzed coplanar polychlorinated biphenyls (PCBs) and polychlorinated-dibenzo-p-dioxins (PCDDs) in human saliva as compared with blood specimens, and examined their effects on human gingival epithelial cells (HGEC). High levels of tri- and tetrachlorinated PCBs were found in saliva, whereas we detected predominantly hexa- and heptachlorinated PCBs in blood. Among PCDDs, the saliva and blood specimens contained mainly 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD). Among the toxic dioxins proposed by the World Health Organization (WHO) in 1998, 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118) and OCDD, which were mainly found in saliva, significantly induced IL-8 production in HGEC. Furthermore, these two dioxins markedly augmented IL-8 production stimulated with fimbriae from Porphyromonas gingivalis, which is well-known as a pathogenic factor in periodontal diseases. These results suggest that dioxins in saliva may be a risk factor for periodontal diseases.