Excessive fluoride intake alters the MMP-2, TIMP-1 and TGF-ß levels of periodontal soft tissues: an experimental study in rabbits.

OBJECTIVES: This study evaluated the influence of fluoride on periodontal soft tissues by investigating any alterations in their MMP-2, TIMP-1 and TGF-ß profiles secondary to excessive fluoride intake. MATERIAL AND METHODS: Fluorosis was induced in 18 rabbits (test group) through consumption of fluoride added to drinking water, whereas 10 rabbits consumed regular tap water as daily supply (control group). Following fluorosis verification, animals were sacrificed and their 1st mandibular molar teeth were utilized in the assessments. MMP-2, TIMP-1 and TGF-ß were separately investigated for gingival epithelium (GE), gingival connective tissue (GC) and periodontal ligament (PL) to evaluate periodontal soft tissues. Histological sections were prepared from the groups, the parameters were determined by immunohistochemistry, and their levels were calculated by quantification of the immunostainings. RESULTS: Staining intensity of MMP-2 in GC and PL (p < 0.01); TIMP-1 and TGF-ß of GE, GC and PL (p < 0.01) were higher in the test group compared to those of the control group. Intra-group staining of TIMP-1 was higher than MMP-2 in all test group compartments (p < 0.01) and in the control group GE (p < 0.01). TIMP-1 was also higher than TGF-ß in the GE and PL of the test group (p < 0.05) and in the GE of the control group (p < 0.01). CONCLUSION: These results suggest that excessive fluoride intake may affect periodontal soft tissues by increasing MMP-2, TIMP-1 and TGF-ß, and thereby altering the MMP-2/TIMP-1 and TIMP-1/TGF-ß ratios. CLINICAL RELEVANCE: Excessive fluoride consumption may alter the periodontal tissue homeostasis which may be detrimental in the maintenance of periodontal health.

Mast cells and MMP-9 in the lamina propria during eruption of rat molars: quantitative and immunohistochemical evaluation.

During the active tooth eruption process, structural changes in the lamina propria are necessary to provide extracellular matrix remodelling and for the establishment of the eruptive pathway. A large number of resident cells, recruited cells and proteases have been demonstrated in the eruptive process, but the participation of MMP-9 and mast cells has not yet been demonstrated. In this study, we set out to evaluate the intensity of MMP-9 immunoexpression, the frequency of mast cells and the correlation between the incidence of mast cells and bone resorption in different phases of tooth eruption. Fragments of maxilla containing first molars, obtained from 9-, 11-, 13- and 16-day-old rats, were fixed in 4% formaldehyde, decalcified and embedded in paraffin. Sagittal sections were stained with Masson's trichrome or submitted to the tartrate-resistant acid phosphatase method for quantification of osteoclasts. Sections stained by 1% toluidine blue were used for quantification of metachromatic mast cells mm(-2) of lamina propria. The expression of MMP-9 in the lamina propria was evaluated by immunohistochemistry. In the 9-day-old rats, the lamina propria contained few mast cells and occasional osteoclasts were found in the bone surface overlying the occlusal portion of the tooth germs. Otherwise, a significant increase in the number of mast cells was observed in the intra-osseous phase of tooth eruption (11-day-old rats), period in which numerous TRAP-positive osteoclasts were found in the bone surface. MMP-9 immunolabelling was detected in fibroblasts, mast cells and macrophage-like cells of the lamina propria in all ages studied. However, an enhanced immunolabelling was evident in the advanced phase of tooth eruption (16-day-old rats). During the intra-osseous phase, the parallel between the high frequency of both mast cells and osteoclasts suggests that mast cells could exert a paracrine function on the osteoclasts and then stimulate bone resorption. The immunoexpression of MMP-9 in different cells of lamina propria, including mast cells, indicates that this enzyme participates in the degradation of ECM, mainly during late phase of mucosal penetration. Thus mast cells and MMP-9 are involved in the complex process of degradation of the eruptive pathway extracellular matrix.

Differential gene expression profiling of the molar tooth germ in peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and in wild-type mouse: molar tooth phenotype of PPAR-alpha knockout mouse.

Gene expression profiling of the first molar tooth germ at embryonic days (E)17.5 and 18.5, and at postnatal days (P)0, 2, and 6 from peroxisome proliferator-activated receptor-alpha (PPAR-alpha) knockout mouse and from wild-type mouse was carried out using microarrays and validated using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. When comparing expression profiles at each time-point, a total of 1,235 genes showed significantly different expression, 772 of which exhibited significantly decreased expression in tooth germ from knockout mouse. With genes exhibiting significantly decreased levels of expression in tooth germ from PPAR-alpha knockout mouse, bioinformatic analysis using ingenuity pathway analysis yielded significant associations to cellular functions related to cellular growth/proliferation and to networks related to regulation of calcium homeostasis. Using scanning electron microscopy to investigate molars from adult PPAR-alpha knockout mouse, the molar size was found to be slightly reduced, the enamel structure was found to be normal, but cervical molar enamel exhibited evidence suggesting hypomineralization. Although the PPAR-alpha knockout had no significant effect on molar morphology, the results suggest that active PPAR-alpha signaling is required to achieve normal mineralization of molar enamel, most probably through regulation of calcium homeostasis and metabolism of vitamin D. Cyp27b1 was expressed in tooth germ, suggesting that tooth germ can synthesize active vitamin D. Expression of Cyp27b1 was significantly enhanced in postnatal PPAR-alpha knockout tooth germ.

Expression of nitric oxide synthases in orthodontic tooth movement.

OBJECTIVE: To investigate differential expression of NOS isoforms in periodontal ligament (PDL) and bone in tension and pressure sides using a rat model of orthodontic tooth movement (OTM). MATERIALS AND METHODS: Immunohistochemistry with NOS isoform (iNOS, eNOS, and nNOS) antibodies was performed on horizontal sections of the first maxillary molars subjected to 3 and 24 hours of OTM. The PDL and adjacent osteocytes of the distopalatal root at pressure and tension areas were analyzed for expression of these proteins. The contralateral molar served as a control. Results were analyzed with one-way ANOVA and with two-way ANOVA. RESULTS: Expression of all isoforms was increased in the tension side. iNOS and nNOS expression in the pressure side with cell-free zone was decreased but in the pressure side without cell-free zone was increased. The number of eNOS-positive cells did not change, but the intensity of the staining was visibly increased in the tension side. Duration of OTM changed only the pattern of nNOS expression. Osteocyte NOS expression did not change significantly in response to OTM. CONCLUSIONS: All NOS isoforms are involved in OTM with different expression patterns between tension and pressure sides, with nNOS being more involved in early OTM events. NOS expression did not change in osteocytes, suggesting that PDL cells rather than osteocytes are the mechanosensors in early OTM events with regard to NO signaling.

Degradation of noncollagenous components by neutrophil elastase reduces the mechanical strength of rat periodontal ligament.

BACKGROUND AND OBJECTIVE: We have previously shown that increases in neutrophil elastase in periodontal ligament with chronic periodontitis results in degradation of the noncollagenous components. The purpose of this study was to investigate whether the destruction of noncollagenous components by treatment with elastase in vitro causes changes in the mechanical properties of the periodontal ligament. MATERIAL AND METHODS: The transverse sections of mandibular first molars, prepared from male Wistar rats at 6 wk of age, were digested with 0-50 microg/mL of neutrophil elastase at 37 degrees C for 4 h. Then, their mechanical properties and morphological features were examined. RESULTS: Digestion with elastase dose-dependently decreased the maximum shear stress and failure strain energy density of the periodontal ligament (p < 0.05-0.01). The histological observations after digestion revealed marked degradation of oxytalan fibers, but no marked changes of the collagen fibers, which was confirmed by the detection of very low quantities of hydroxyproline in the digest. The light and scanning electron micrographs showed that the elastase degraded the interfibrillar substances in the periodontal ligament and exposed individual collagen fibrils. CONCLUSION: These results suggest that the increased neutrophil elastase observed in periodontal disease degrades the oxytalan fibers and interfibrillar substances in the periodontal ligament to decrease its mechanical strength.

Evaluation of root resorption associated with orthodontic movement in stressed rats.

AIM: The aim of this study was to investigate the effects of acute and chronic systemic stress response on orthodontically induced root resorption. METHODS: Male Wistar rats were restrained during 1 hour a day by stress models of short (3 days) and long duration (40 days), while control group was not submitted to restraint (N.=10/group). The upper left first molars of all rats were moved mesially by a fixed orthodontic appliance exerting 50 g force upon insertion during the last 14 days of the experiment. Then, animals were killed for blood collection and mensuration of plasmatic corticosterone by radioimmunoassay; the tissues around mesial root of the first molar were processed for histological and histochemical techniques with tartrate-resistant acid phosphatase. The degree of root resorption and the number of odontoclasts were evaluated, being the contralateral side of each animal serving as its control (split-mouth design). RESULTS: The results revealed that the plasmatic levels of corticosterone were significantly higher in both the stressed groups than in the control one. There were no significant differences in the degree of root resorption and in the number of odontoclasts on the root between the 3 groups studied. CONCLUSIONS: These results indicate that systemic stress alone can not be considered a risk factor for root resorption induced by orthodontic tooth movement.

MMP20 Overexpression Disrupts Molar Ameloblast Polarity and Migration.

Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20+/+Tg+) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20+/+Tg+ mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted ß-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the ß-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20+/+Tg+ molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20+/+Tg+ mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.

Role of the friction free distalize appliance (2FDA)PAT in the molar distalization: photoelastic analysis and alkaline-phosphatase (ALP) activity on first molar and bicuspid.

Maxillary molar distalization is an increasingly popular option for the resolution of Class II malocclusions. This study describes the effects of one particular molar distalizing appliance, the Friction Free Distalize Appliance (2FDA), in a sample of 20 consecutively treated and growing patients to verify the osteoblastic activity in the compression and traction sites of both the molars and the bicuspids when used as the anchorage teeth. The 2FDA appliances were constructed utilizing a Nickel Titanium open coil spring of 200 gr force in order to distalize the maxillary first molar. The reaction force was controlled utilizing the principle of low/free friction. The results show that the resin around the root of the bicuspid did not discolour at all, which indicates an absence of a force load. On the other hand, on the molar, the resin around the root of the molar became discoloured due to the fact that an orthodontic force was involved with the tooth. To better understand whether the quantity of force that reached the tooth was able to produce osteoblastic recruitment in the sites of tension of the molar and the bicuspid, we quantified an enzyme, the alkaline phosphatase (ALP), present. This measurement allowed us to verify a regular increase of the ALP on the site of molar traction. We also elaborated a mathematical model to evaluate the quantity of force of reaction that produces the device on the bicuspid. Such force results as being 8.34 grams which equals half the pressure of the capillaries of the parodontal ligament (18 grams). The 2FDA appliance compares favourably with other intra-oral distalization devices for the resolution of patients with Class II malocclusions, and is the only distalizing appliance that does not determine osteoclastic/osteoblastic recruitment on the anchorage tooth.

Molar tooth development in caspase-3 deficient mice.

Tooth morphogenesis is accompanied by apoptotic events which show restricted temporospatial patterns suggesting multiple roles in odontogenesis. Dental apoptosis seems to be caspase dependent and caspase-3 has been shown to be activated during dental apoptosis.Caspase-3 mutant mice on different genetic backgrounds were used to investigate alterations in dental apoptosis and molar tooth morphogenesis. Mouse embryos at E15.5 were analyzed to reveal any changes in enamel knots, which are transient structures eliminated by apoptosis. In caspase-3(-/-) mice on the B57BL/6 background, disorganization of the epithelium was found in the original primary enamel knot area and confirmed by altered expression of Shh. Despite this early defect in molar tooth development, these mutants showed correct formation of secondary enamel knots as indicated by Fgf-4 expression. Analyses of adult molar teeth did not reveal any major alterations in tooth shape, enamel structure or pattern when compared to heterozygote littermates. In caspase-3(-/-) mice on the 129X1/SvJ background, no defects in tooth development were found except the position of the upper molars which developed more posteriorly in the oral cavity. This is likely, however, to be a secondary defect caused by a physical squashing of the face by the malformed brain. The results suggest that although caspase-3 becomes activated and may be essential for dental apoptosis, it does not seem fundamental for formation of normal mineralised molar teeth.

Origin, splicing, and expression of rodent amelogenin exon 8.

Amelogenin RNA transcripts undergo extensive alternative splicing, and MMP-20 processes the isoforms following their secretion. Since amelogenins have been ascribed cell-signaling activities, we asked if a lack of proteolytic processing by MMP-20 affects amelogenin signaling and consequently alters amelogenin splice site selection. RT-PCR analyses of amelogenin mRNA between control and Mmp20(-/-)mice revealed no differences in the splicing pattern. We characterized 3 previously unidentified amelogenin alternatively spliced transcripts and demonstrated that exon-8-encoded amelogenin isoforms are processed by MMP-20. Transcripts with exon 8 were expressed approximately five-fold less than those with exon 7. Analyses of the mouse and rat amelogenin gene structures confirmed that exon 8 arose in a duplication of exons 4 through 5, with translocation of the copy downstream of exon 7. No downstream genomic sequences homologous to exons 4-5 were present in the bovine or human amelogenin genes, suggesting that this translocation occurred only in rodents.

NO-cGMP signaling molecules in cells of the rat molar dentin-pulp complex.

By the formation of cyclic guanosine 3',5'-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC alpha(2)-subunit/beta(1)-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC alpha(2), and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10(-5) M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10(-4) M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.

Carbonic anhydrase in developing hamster molars.

Carbonic anhydrase (CA) activity decreased slightly in hamster molars from one to four d after birth. It was inhibited by low concentrations of acetazolamide. Histochemically, the stratum intermedium and stellate reticulum showed an intense staining for CA, whereas ameloblasts increased and odontoblasts decreased their CA activity with age.

Cloning, characterization, and expression analysis of mouse enamelysin.

Enamelysin is a recently isolated member of the matrix metalloproteinase (MMP) family of extracellular matrix (ECM)-degrading enzymes. Here we describe the isolation and characterization of the mouse enamelysin cDNA. Expression of mouse enamelysin was detectable only in ameloblasts and odontoblasts of developing teeth. Characterization of mouse enamelysin demonstrated that it is highly conserved in both its sequence content and pattern of expression relative to the porcine, human, and bovine homologues previously described.

Influence of various inhibitors on alkaline phosphatase in calicifying hamster molars, employing paranitrophenyl phosphate as substrate.

In mineralizing hamster molars, the influence of several inhibitors on p-NPP-ase has been demonstrated, and compared with their effect on PPi-ase, known to belong to the same enzyme. Whereas the latter activity is much more physiological, p-NPP is a better substrate to distinguish between the effects of bivalent cations on the enzyme or the substrate.

Localization of EMSP1 expression during tooth formation and cloning of mouse cDNA.

Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.

Separation and partial purification of acid phosphates of the enamel organ of rat molars.

At least two types of acid phosphatases with markedly different properties were separated from the enamel organ of rat molar tooth buds. One enzyme (A) bound weakly to the CM-cellulose column and was eluted with a combined linear salt and pH gradient; another enzyme (B) bound strongly to the column and was eluted with a second linear salt gradient at constant pH. Enzyme A was identified as a phosphomonoester hydrolase (3.1.3.2) similar to the lysosomal enzyme of soft tissues and the tartrate-sensitive enzyme of bone. Enzyme B did not hydrolyse aliphatic monophosphate ester substrates but, like enzyme A, it did split the aryl monophosphate ester substrate, para-nitrophenylphosphate, as well as the phosphate esters of casein and the acid anhydride substrates, ATP and inorganic pyrophosphate. This enzyme is similar to the low molecular weight tartrate-resistant acid phosphatases of bone and soft tissues.

The matrix metalloproteinase gelatinase A in human dentine.

A dentine protein extraction protocol was modified in order to identify matrix metalloproteinase gelatinolytic activities in the non-mineralized and mineralized phases of human dentine. Dentine proteins from 24 individual permanent molars from patients aged 15-73 years were sequentially extracted, first with guanidinium chloride (G1 extract), then EDTA (E extract), and after this demineralization step, again by guanidinium chloride (G2 extract) to dissociate collagen-associated proteins. Extracts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and the gels were processed by Western blotting and zymography to detect gelatinolytic activities. Active and latent forms of gelatinase A were identified in the non-mineralized dentine fraction (G1 extract) of 58% of the teeth. Other gelatinolytic species were also detected by zymography with apparent M(r) of 92, 54 and 30 kDa. Although gelatinase A was detected in the G1 extracts of teeth from all ages, indicating more recent synthesis and remodelling of the predentine, gelatinase A was never detected in any E extract or in the G2 extracts of patients older than 41 years. The presence of the active form of gelatinase A in mineralized human dentine implicates this enzyme in dentine mineralization.

Aryl sulfatase activity in mouse molar odontogenesis.

Arylsulfatase activity has been studied in the developing molar of the Swiss albino mouse from the lamina stage to the appositional stage. Timed-pregnant Swiss albino mice were utilized in this study. Females were sacrificed by ether anesthesia and fetuses extirpated or newborns anesthetized and decapitated. Frozen sections were fixed and incubated for arylsulfatase activity according to a modification of the method of PEARSE (1972). The tissue was dehydrated, cleared and covered. Phase light microscopy was utilized in evaluating arylsulfatase activity in the developing molar. Arylsulfatase activity was evaluated for each stage of development and the results presented in tabular form. The present investigation represents the first known effort to describe arylsulfatase activity in odontogenic tissues from the initiation of the dental lamina through the appositional stage. Arylsulfatase activity appeared to be related to the degree of vascularization of the developing enamel organ and adnexa and the beginning of hard tissue elaboration.

Thiamine pyrophosphatase activity of the prenatal mouse molar. A histochemical investigation.

Thiamine pyrophosphatase (TPPase), an enzyme associated with the Golgi apparatus, has been implicated in the regulation of cellular oxidation as well as of transport across cell membranes. This enzyme has been localized in odontogenic tissues of the postnatal mouse and it was the intent of the present study to localize TPPase during prenatal odontogenesis. Mouse fetuses (CDI, Charles River) 14 through 19 days postconception were decapitated, the heads were frozen and mounted on the chuck of a cryostat. Frontal sections, 14 micrometers thick, were air-dried and incubated for TPPase activity. Subsequent to incubation the activity was visualized by immersion in 1% ammonium sulfide. The degree of enzyme activity varied not only with the chronological age of the fetus but also as a function of the tissue's metabolic state. Regions, such as the dental lamina, evidenced decreased TPPase activity with increasing age, while tissue layers such as the IEE displayed greater enzyme activity with increasing age.

Cytochemical localization of adenylate-cyclase in embryonic mouse molars.

The cytochemical method of HOWELL and WHITFIELD (1972) and the revised cytochemical medium of KEMPEN et al. (1978) has been used to investigate the localization of adenylate-cyclase activity in 18, 19 and 20 day old first embryonic mandibular molars. Membrane associated precipitate granules were observed in preameloblasts and odontoblasts localized in the area where preodontoblasts become post-mitotic. Heat inactived teeth and the samples incubated in media without substrate were negative for membrane bound lead deposits. A correlation may exist between high adenylate-cyclase activity and the terminal differentiation of odontoblasts.