p38α MAPK is required for tooth morphogenesis and enamel secretion.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and ß4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.

Analysis of bone formation after sinus augmentation using ß-tricalcium phosphate.

Implant placement in the edentulous maxilla often represents a clinical challenge due to insufficient bone height after crestal bone resorption and maxillary sinus pneumatization. Several graft materials have been evaluated for augmenting the maxillary sinus to compensate for the lost vertical dimension. Allografts are readily available without the risk of disease transmission and the need for a second site surgery. The aim of this case series was to systematically evaluate the development and maturation of augmented bone in the maxillary sinus using beta-tricalcium phosphate. In 21 to 40 weeks post-sinus elevation, bone biopsies were taken and implants placed simultaneously. All specimens were demineralized and subjected to staining procedures (ie, Hematoxylin and Eosin [H&E], Goldner's staining, and tartrate-resistant acid phosphatase [TRAP]). Total bone increased over time, whereas the amount of graft material diminished. A lack of inflammatory reaction was noticed with the use of this graft material. In addition, TRAP staining revealed the presence of osteoclasts surrounding the remaining particles. During a 12-month follow-up, no implant failure or complications were observed.

Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino-enamel junction, and a decussating enamel rod pattern.

Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.

Tbx1 regulates progenitor cell proliferation in the dental epithelium by modulating Pitx2 activation of p21.

Tbx1(-/-) mice present with phenotypic effects observed in DiGeorge syndrome patients however, the molecular mechanisms of Tbx1 regulating craniofacial and tooth development are unclear. Analyses of the Tbx1 null mice reveal incisor microdontia, small cervical loops and BrdU labeling reveals a defect in epithelial cell proliferation. Furthermore, Tbx1 null mice molars are lacking normal cusp morphology. Interestingly, p21 (associated with cell cycle arrest) is up regulated in the dental epithelium of Tbx1(-/-) embryos. These data suggest that Tbx1 inhibits p21 expression to allow for cell proliferation in the dental epithelial cervical loop, however Tbx1 does not directly regulate p21 expression. A new molecular mechanism has been identified where Tbx1 inhibits Pitx2 transcriptional activity and decreases the expression of Pitx2 target genes, p21, Lef-1 and Pitx2c. p21 protein is increased in PITX2C transgenic mouse embryo fibroblasts (MEF) and chromatin immunoprecipitation assays demonstrate endogenous Pitx2 binding to the p21 promoter. Tbx1 attenuates PITX2 activation of endogenous p21 expression and Tbx1 null MEFs reveal increased Pitx2a and activation of Pitx2c isoform expression. Tbx1 physically interacts with the PITX2 C-terminus and represses PITX2 transcriptional activation of the p21, LEF-1, and Pitx2c promoters. Tbx1(-/+)/Pitx2(-/+) double heterozygous mice present with an extra premolar-like tooth revealing a genetic interaction between these factors. The ability of Tbx1 to repress PITX2 activation of p21 may promote cell proliferation. In addition, PITX2 regulation of p21 reveals a new role for PITX2 in repressing cell proliferation. These data demonstrate new functional mechanisms for Tbx1 in tooth morphogenesis and provide a molecular basis for craniofacial defects in DiGeorge syndrome patients.

Targeted p120-catenin ablation disrupts dental enamel development.

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows.

The acid test of fluoride: how pH modulates toxicity.

BACKGROUND: It is not known why the ameloblasts responsible for dental enamel formation are uniquely sensitive to fluoride (F(-)). Herein, we present a novel theory with supporting data to show that the low pH environment of maturating stage ameloblasts enhances their sensitivity to a given dose of F(-). Enamel formation is initiated in a neutral pH environment (secretory stage); however, the pH can fall to below 6.0 as most of the mineral precipitates (maturation stage). Low pH can facilitate entry of F(-) into cells. Here, we asked if F(-) was more toxic at low pH, as measured by increased cell stress and decreased cell function. METHODOLOGY/PRINCIPAL FINDINGS: Treatment of ameloblast-derived LS8 cells with F(-) at low pH reduced the threshold dose of F(-) required to phosphorylate stress-related proteins, PERK, eIF2alpha, JNK and c-jun. To assess protein secretion, LS8 cells were stably transduced with a secreted reporter, Gaussia luciferase, and secretion was quantified as a function of F(-) dose and pH. Luciferase secretion significantly decreased within 2 hr of F(-) treatment at low pH versus neutral pH, indicating increased functional toxicity. Rats given 100 ppm F(-) in their drinking water exhibited increased stress-mediated phosphorylation of eIF2alpha in maturation stage ameloblasts (pH<6.0) as compared to secretory stage ameloblasts (pH approximately 7.2). Intriguingly, F(-)-treated rats demonstrated a striking decrease in transcripts expressed during the maturation stage of enamel development (Klk4 and Amtn). In contrast, the expression of secretory stage genes, AmelX, Ambn, Enam and Mmp20, was unaffected. CONCLUSIONS: The low pH environment of maturation stage ameloblasts facilitates the uptake of F(-), causing increased cell stress that compromises ameloblast function, resulting in dental fluorosis.

Nanoparticle-based endodontic antimicrobial photodynamic therapy.

OBJECTIVE: To study the in vitro effects of poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with the photosensitizer methylene blue (MB) and light against Enterococcus faecalis (ATCC 29212). MATERIALS AND METHODS: The uptake and distribution of nanoparticles in E. faecalis in suspension was investigated by transmission electron microscopy (TEM) after incubation with PLGA complexed with colloidal gold particles for 2.5, 5, and 10 minutes. E. faecalis species were sensitized in planktonic phase and in experimentally infected root canals of human extracted teeth with MB-loaded nanoparticles for 10 minutes followed by exposure to red light at 665 nm. RESULTS: The nanoparticles were found to be concentrated mainly on the cell walls of microorganisms at all three time points. The synergism of light and MB-loaded nanoparticles led to approximately 2 and 1 log10 reduction of colony-forming units (CFUs) in planktonic phase and root canals, respectively. In both cases, mean log10 CFU levels were significantly lower than controls and MB-loaded nanoparticles without light. CONCLUSION: The utilization of PLGA nanoparticles encapsulated with photoactive drugs may be a promising adjunct in antimicrobial endodontic treatment.

Guinea pig model of Mycobacterium tuberculosis latent/dormant infection.

Although guinea pigs are considered one of the best animal models of tuberculosis, little data exist describing latent or dormant tuberculosis infection in these animals. Here we address this issue using a streptomycin auxotrophic mutant of Mycobacterium tuberculosis. This mutant grows unimpaired in the presence of streptomycin but in its absence shifts to latency/dormancy (lack growth and over-expression of alpha-crystallin). To establish infection animals are inoculated with the mutant followed by daily administration of streptomycin (three weeks), which allows initial microbial multiplication in the animal's tissues. Withdrawal of streptomycin establishes latency/dormancy and few viable organisms are recovered from the animals' lungs and spleen six months later. During the infectious process guinea pigs steadily gained weight and presented no clinical signs (scuff fur and lethargy) of disease. Histopathology of organs mimicked tuberculous lesions in humans and PBMC from infected animals strongly responded to stimulation with PPD. Finally, tuberculin skin test (a hallmark of latent infection diagnosis) performed in infected animals was strongly positive (>or=15 mm induration). These results point to an interesting and reliable model of latent/dormant tuberculosis infection in guinea pigs.

A developmental comparison of matrix metalloproteinase-20 and amelogenin null mouse enamel.

Mutations in both the human amelogenin and human matrix metalloproteinase-20 (MMP20, enamelysin) genes cause amelogenesis imperfecta. Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage-specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP-20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP-20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate-like, while the MMP-20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid-secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP-20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full-thickness enamel containing the characteristic decussating prism pattern.

Formation of the dentino-enamel interface in enamelysin (MMP-20)-deficient mouse incisors.

An anomalous dentino-enamel junction (DEJ), manifested by delamination of the enamel layer, was reported in enamelysin [matrix metalloproteinase-20 (MMP-20)] knockout (KO) mice. To better understand the possible role of MMP-20 in the formation of the DEJ, we performed transmission electron microscopy (TEM) studies of the DEJ at early stages of tooth morphogenesis in KO mice. Our TEM analysis revealed that in the incisors from KO mice the mantle dentin is hypomineralized at the onset of enamel mineralization. At this early stage, TEM revealed no apparent differences in nascent aprismatic enamel between the KO mice and the controls. Hypomineralized mantle dentin was also observed in the incisors from KO mice, as assessed by back-scattered SEM at the secretory and early maturation stages, but not in the late-maturation stage, suggesting that the mineralization of mantle dentin is not completely arrested, but rather postponed. Histological studies indicate that the organic content in the initial enamel layer remains very high throughout amelogenesis. These results imply that MMP-20 is involved in the regulation of mineralization in mantle dentin and demonstrate the complex nature of DEJ formation. They also suggest that the structural and functional properties of the DEJ are determined during the initial mineralization stages.

Overexpression of transforming growth factor-beta1 in teeth results in detachment of ameloblasts and enamel defects.

Transforming growth factor-beta1 (TGF-beta1) is a key regulator of many cellular processes, including cell adhesion, the immune response and synthesis of extracellular matrix proteins. In the present study, we report the characterization of enamel defects in a transgenic mouse model overexpressing TGF-beta1 in odontoblasts and ameloblasts, its expression being driven by the promoter sequences of the dentin sialophosphoprotein gene. As reported earlier, these mice develop distinct dentin defects similar to those seen in human dentin dysplasia and dentinogenesis imperfecta. A further detailed examination of enamel in these mice revealed that from the early secretory stage, ameloblasts began to detach from dentin to form cyst-like structures. A soft X-ray analysis revealed that this cyst-like structure had a disorganized and partially mineralized matrix with an abnormal mineralization pattern and a globular appearance. In the molars, the enamel was not only pitted and hypoplastic, but enamel rods were completely lost. Thus, altered TGF-beta1 expression in the tooth seems to trigger detachment of ameloblasts and abnormal secretion and deposition of minerals in the cyst-like structures adjoining the dentin. We speculate that the altered expression of TGF-beta1 in teeth impacts the adhesion process of ameloblasts to dentin.

SEM evidence that one ameloblast secretes one keyhole-shaped enamel rod in monkey teeth.

Primate enamel is subdivided into inner enamel, having Hunter-Schreger bands, and outer enamel with all rods parallel to each other. Outer inter-rod enamel may surround each rod, lie between rows of rods, or be absent, as in the 'keyhole pattern', which is composed entirely of rods. One theory on the formation of the 'keyhole' pattern overlays the hexagonal cross-sectional shape of four or more ameloblasts over the keyhole shape of the enamel rod. This ignores the likelihood that Tomes processes have a different shape from the cell body, and also ignores the observation that paths of enamel rods sometimes diverge. Scanning electron microscopy (SEM) revealed the keyhole shape of the forming face of monkey enamel. These forming rods were arranged in stepped rows with the head regions in each row separated by the tails of the preceding row. Consequently, each forming face of a rod was surrounded on three sides by previously formed enamel. The apical surface of the Tomes process was shaped exactly like the forming rod face, permitting direct apposition of one rod and one Tomes process. The conclusion was that, in the monkey, each rod of the keyhole enamel configuration is produced by one ameloblast.

In vitro evaluation of the implant-abutment bacterial seal: the locking taper system.

PURPOSE: To test in vitro whether the seal provided by the locking taper used in the implant-abutment connection was capable of preventing the invasion of oral microorganisms. MATERIALS AND METHODS: Twenty-five wide-body implants (5 x 11 mm) and 25 abutments were divided into 2 groups for a 2-phase experiment. The first phase tested the ability of the seal to shield the implant well from outside bacteria; the second phase tested the ability of the seal to prevent bacteria present in the implant well from seeping out. For phase 1, 10 implant-abutment units were immersed in a bacterial broth for 24 hours. The abutments were then separated from the implants and bacterial presence was evaluated using scanning electron microscopy. In phase 2, the tested abutments were inoculated with a droplet of soft agar bacterial gel and assembled with the implant. These units were incubated in a sterile nutrient broth for 72 hours, sampled, and plated to assess bacterial presence. RESULTS: In phase 1, no bacteria were detected in any of the implant wells. In phase 2, no bacteria were detected in the nutrient broth or on the agar plates at 72 hours. DISCUSSION: In implants where a microgap is present, microbial leakage could lead to inflammation and bone loss; thus, it is important to minimize bacterial presence in and around the the implant-abutment junction. CONCLUSION: The seal provided by the locking taper design has been demonstrated to be hermetic with regard to bacterial invasion in vitro.

Comparison of the effectiveness of scaling and root planing in vivo using hand vs rotary instruments.

The purpose of the present investigation was to compare the effectiveness of a scaling bur and conventional Gracey curettes in vivo. Ten teeth scheduled for extraction were scaled and root planed thoroughly before extraction. The instrumented areas were observed using SEM. Scaling with a No. 12 fluted carbide bur was more effective in removing debris and plaque than were conventional Gracey curettes. Because of the aggressive nature of the process, a certain learning curve is necessary to get accustomed to bur scaling; otherwise, dentinal hypersensitivity and patient discomfort may be increased.

Evaluation of the whole genome DNA-DNA hybridization technique to identify bacteria in histological sections of periradicular lesions.

Whole genome DNA-DNA hybridization has been used to identify bacteria in periradicular lesions partly because there is no amplification of the bacteria, therefore, minor contaminants are not detected. There are, however, potential pitfalls with this technique, including inability to distinguish dead bacteria, cross-reactions of species within a genus, and inability to detect species present in low numbers because of loss of DNA during extraction and purification. Alternatively, inadequate extraction and purification of DNA could result in false positives. Therefore, controls are required to monitor DNA loss, DNA cross-reactions, and DNA of pure cultures mixed with bacteria-free tissue to monitor for false positives. We determined that the quality of DNA extracted from histological sections of periradicular lesions is excellent for DNA-DNA hybridization. Although lesions contain large numbers of bacteria, histological sections through lesions barely contain sufficient quantity of bacteria for such analysis. This was confirmed by histological observation of sparsely distributed bacteria within lesions. Furthermore, we found that the bacteria are not distributed evenly throughout periradicular lesions, in numbers or species.

Delayed tooth eruption in membrane type-1 matrix metalloproteinase deficient mice.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed highly in mineralizing tissues including bones and teeth. Mice deficient in MT1-MMP (-/-) display severe defects in skeletal development including dwarfism, osteopenia, and craniofacial abnormalities. Death occurs in these mice by about 3 weeks of age. Since MT1-MMP is expressed by the ameloblasts of the enamel organ and by the odontoblasts of the dental papilla, we asked if the developing teeth were adversely affected in the knockout animals. Molars from MT1-MMP -/- mice and controls were examined by histological, X-ray, and SEM analysis at 4, 18-20, and 25 days of postnatal development. At 4 days of development the molars from the -/- mice appeared histologically normal. At 18-20 days of development, the first molars of the -/- mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. In contrast to the -/- mice, the first molars of the 18-20-day control animals had erupted. SEM analysis of a -/- first molar and incisor revealed a normal enamel prism pattern. However, X-ray analysis demonstrated that tooth eruption was delayed by approximately 5 days and that the tooth roots were abnormally short in the knockout animals. Since MT1-MMP-deficient mice have been demonstrated to display a generalized increase in bone resorption, these data suggest that inefficient growth of bone surrounding the tooth root complex causes a delay in tooth eruption.

Enamelysin (matrix metalloproteinase 20)-deficient mice display an amelogenesis imperfecta phenotype.

Enamelysin is a tooth-specific matrix metalloproteinase that is expressed during the early through middle stages of enamel development. The enamel matrix proteins amelogenin, ameloblastin, and enamelin are also expressed during this same approximate developmental time period, suggesting that enamelysin may play a role in their hydrolysis. In support of this interpretation, recombinant enamelysin was previously demonstrated to cleave recombinant amelogenin at virtually all of the precise sites known to occur in vivo. Thus, enamelysin is likely an important amelogenin-processing enzyme. To characterize the in vivo biological role of enamelysin during tooth development, we generated an enamelysin-deficient mouse by gene targeting. Although mice heterozygous for the mutation have no apparent phenotype, the enamelysin null mouse has a severe and profound tooth phenotype. Specifically, the null mouse does not process amelogenin properly, possesses an altered enamel matrix and rod pattern, has hypoplastic enamel that delaminates from the dentin, and has a deteriorating enamel organ morphology as development progresses. Our findings demonstrate that enamelysin activity is essential for proper enamel development.

Ultrastructure of the dental epithelium and odontoblasts during enameloid matrix deposition in cichlid teeth.

Teleost enameloid matrix has been proposed to be an ectodermal, mesodermal, or joint ectodermal-mesodermal product. To determine its origin we examined the ultrastructure of the inner dental epithelium (IDE), odontoblasts, enameloid, and dentin matrices of cichlid tooth buds at the stage of enameloid formation. © Alan R. Liss, Inc. Columnar IDE cells had apical and basal terminal webs and contained organelles associated with protein synthesis, including elongated secretory granules containing fibrillar material having cross-striations with 60-nm periodicity. The morphology of IDE secretory granules was typical of procollagen granules observed in a large variety of ectodermal and mesodermal cells synthesizing collagen. In contrast, the paucity of secretory granules within three odontoblast types indicates that these cells probably do not synthesize enameloid matrix. These observations are consistent with the idea that the bulk of the enameloid matrix is itself an ectodermal collagen synthesized and secreted by IDE cells.