Dental staining after doxycycline use in children.

Background: The use of doxycycline has been avoided before 8 years of age due to known dental staining caused by tetracyclines, although doxycycline differs from classical tetracyclines in many ways. Doxycycline is still an important antimicrobial agent, but its dental safety is not well studied. Objectives: To examine the state of permanent teeth after doxycycline exposure in children <8 years of age. Methods: Details of doxycycline treatment were collected from medical records. After the eruption of permanent teeth the dental status was examined by an experienced paediatric dentist for detection of dental staining and enamel hypoplasia. The resulting dental photographs were evaluated by a second independent experienced paediatric dentist. Results: The mean age of 38 study subjects at the time of doxycycline treatment was 4.7 years (range 0.6-7.9 years, SD 2.3). The doxycycline dose was 10 mg/kg/day (varying from 8 to 10 mg/kg/day) for the first 2-3 days and 5 mg/kg/day (varying from 2.5 to 10 mg/kg/day) thereafter. The mean length of the treatment was 12.5 days (SD 6.0) and ranged from 2 to 28 days. Tetracycline-like staining or enamel hypoplasia of developing teeth was detected in none of the subjects. Conclusions: Doxycycline treatment of small children does not seem to induce permanent tooth staining.

Molar-incisor hypomineralization and the association with childhood illnesses and antibiotics in a group of Finnish children.

OBJECTIVE: Molar-incisor hypomineralization (MIH) is a developmental enamel defect affecting 1-4 first permanent molars (FPMs) and often also incisors. The aim of this study was to assess whether childhood illnesses or medication are associated with MIH. MATERIAL AND METHODS: FPMs and incisors of 287 Finnish children were examined for MIH in line with the criteria of the EAPD. Health data from the first 3 years of life was collected from medical records and the associations with MIH and MIH2 (lesions in at least one FPM and incisor) were assessed using simple and multiple logistic regression analyses. RESULTS: The prevalence of MIH and MIH2 were 11.5% and 6.3%, respectively. During the first 3 years of life, the children with MIH had sought care for infectious illnesses more often than the children without MIH (mean number of visits (SD) 7.9(6.4) vs. 6.0(5.1), p = 0.045, independent samples t-test). After adjustment for confounding factors, children who had received penicillin or macrolides within the first year, or amoxicillin within the first 3 years had a higher risk for MIH (2.61, 4.07 and 2.58 times, adjusted OR, respectively) or MIH2 (3.16 times, aOR for penicillin and amoxicillin) compared to those who had not received that antibiotic. Of the illnesses, children with at least one episode of otitis within the first year had a higher risk for MIH (2.28 times, aOR) than those who had not suffered from otitis. CONCLUSIONS: Acute otitis media and the use of certain antibiotics were associated with the elevated risk of MIH/MIH2.

Background factors of molar-incisor hypomineralization in a group of Finnish children.

OBJECTIVE: Molar-Incisor Hypomineralization (MIH) is a common developmental enamel defect characterized by demarcated opacities in permanent molars and incisors. Its etiology still remains unclear. The aim of this retrospective cohort study was to assess if the socioeconomic environment of the child is associated with MIH. MATERIALS AND METHODS: The study was located in two rural towns and three urban cities in Finland. A total of 818 children, between 7-13 years old, were examined for MIH using the evaluation criteria in line with those of the European Academy of Paediatric Dentistry, but excluding opacities smaller than 2 mm in diameter. The mothers filled in a questionnaire which included questions related to the family's way of living (e.g. area of residency, farming, day care attendance) and socioeconomic status (family income, number of mother's school years, level of maternal education). RESULTS: The prevalence of MIH in the study population was 17.1%. Family income, urban residency and day care attendance were associated with MIH in the univariate analysis. In the multivariate analysis using binary logistic regression, only urban residency during a child's first 2 years of life remained associated with MIH. The prevalence of MIH in urban areas was 21.3% and in rural areas 11.5% (OR = 2.18, CI = 1.35-3.53, p = 0.001). CONCLUSIONS: The prevalence of MIH was related to urban residency and could not be explained by any other factor included in the study.

Tributyltin alters osteocalcin, matrix metalloproteinase 20 and dentin sialophosphoprotein gene expression in mineralizing mouse embryonic tooth in vitro.

We showed in a previous in vitro study that tributyltin (TBT) arrests dentin mineralization and enamel formation in developing mouse tooth. The present aim was to investigate the effect of TBT on the expression of genes associated with mineralization of dental hard tissues. Embryonic day 18 mouse mandibular first molars were cultured for 3, 5 or 7 days and exposed to 1.0 µM TBT and studied by real-time quantitative polymerase chain reaction (RT-QPCR) for the expressions of osteocalcin (Ocn), alkaline phosphatase (Alpl), dentin matrix protein 1 (Dmp1), dentin sialophosphoprotein (Dspp) and matrix metalloproteinase 20 (Mmp-20).Ocn, Mmp-20 and Dspp, whose expressions showed changes in RT- QPCR, were further analyzed by in situ hybridization of tissue sections. In situ hybridization showed that TBT decreased Ocn expression in odontoblasts but increased the expression in the epithelial tooth compartment. In QPCR assays, the net effect in the whole tooth was increased expression. TBT also reduced Mmp-20 expression in ameloblasts and odontoblasts. Dspp expression varied but both QPCR assays and in situ hybridization showed a decreasing trend. TBT exposure had no clear effect on Alpl and Dmp1 expressions. Increased Ocn expression by epithelial enamel organ may inhibit dentin mineralization and enamel formation. Decreased Ocn, Mmp-20 and Dspp expressions in odontoblasts may indicate delayed cell differentiation, or TBT may specifically decrease the expression of genes involved in dentin mineralization. While decreased Mmp-20 expression by TBT in ameloblasts may impair enamel mineralization, the coincident reduction in Mmp-20 and Dspp expressions in odontoblasts may potentiate the delay of dentin mineralization.

DLX3 homeodomain mutations cause tricho-dento-osseous syndrome with novel phenotypes.

Tricho-dento-osseous syndrome (TDO) is a rare type of dominantly inherited ectodermal dysplasia so far described only in a few families and associated with 3 known mutations in the DLX3 homeobox gene. Here, we describe two families of Finnish origin that segregate features of TDO in several generations. The affected family members have sparse or curly/kinky hair at birth, markedly delayed or advanced dental maturity, defective tooth enamel and dentin, taurodontic molars, multiple dental abscesses and filling of tooth pulps with amorphous denticle-like material as well as an increased density and/or thickness of craniofacial bones. The disease is especially accentuated in one of the families in which the patients develop only lanugo-type hair and the dental abnormalities are severe. After mutational analysis of DLX3, we identified 2 missense mutations affecting the conserved homeodomain. We suggest that TDO is essentially caused by loss of function and haploinsufficiency of DLX3.

Combined effect of fluoride and 2,3,7,8-tetrachlorodibenzo-p-dioxin on mouse dental hard tissue formation in vitro.

Fluoride interferes with enamel matrix secretion and mineralization and dentin mineralization. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), also impairs dental hard tissue formation and mineralization in vitro and in vivo. Our aim was to investigate in vitro whether the combined effect of sodium fluoride (NaF) and TCDD on dental hard tissue formation is potentiative. For this purpose, mandibular first and second molar tooth germs of E18 mouse embryos were cultured for 5-12 days with NaF and TCDD alone at various concentrations (2.5, 5, 10, 12.5, 15, and 20 µM and 5, 10, 12.5, and 15 nM, respectively) to determine the highest concentrations, which alone cause no or negligible effects. Morphological changes were studied from the whole tooth photographs and histological tissue sections. The concentrations found were 15 µM for NaF and 10 nM for TCDD. While at these concentrations, the effects of NaF and TCDD alone were barely detectable, the effect of simultaneous exposure on dentin and enamel formation was overt; mineralization of predentin to dentin and enamel matrix secretion and mineralization were impaired. Immunohistochemical analysis revealed that the combined exposure modified amelogenin expression by odontoblasts. Morphology of ameloblasts and the expression of amelogenin indicated that ameloblasts were still secretory. The results show that NaF and TCDD have potentiative, harmful effects on the formation of dental hard tissues. Since children can be exposed to subclinical levels of fluoride and dioxins during early childhood, coincidently with mineralization of the first permanent teeth, this finding may have clinical significance.

Isolation and characterization of Streptococcus mitis from blood of child with osteomyelitis.

OBJECTIVES: Osteomyelitis is an inflammatory process accompanied by bone destruction that is caused by bacterial infection, with most child cases showing a haematogenous origin and metaphysis of the long bones. The aim of the present study was to characterize streptococcal strains isolated from the blood of a child diagnosed with osteomyelitis in a long bone and investigate the biological properties related to virulence of strains associated with osteomyelitis. METHODS: Blood isolate species were determined based on the 16S rRNA sequence. Next, the blood isolates were analysed for phagocytosis susceptibility by polymorphonuclear leukocytes, platelet aggregation, inhibitory effects on osteoblastic cells, and their properties of adhesion with cells, and compared to the reference strain Streptococcus mitis ATCC49456. RESULTS: The blood isolates were found to be a single clone (named SA1101), which was determined to be S. mitis. The phagocytosis susceptibility of SA1101 was significantly lower than that of ATCC49456, while its platelet aggregation rate was higher. Furthermore, SA1101 showed an inhibitory effect toward the growth of osteoblastic cells and had greater properties of adhesion to those cells as compared to ATCC49456. CONCLUSIONS: These results suggest that S. mitis SA1101 is a possible etiological agent and caused osteomyelitis in the present case.

The effect of amoxicillin on dental enamel development in vivo.

The exposure to amoxicillin has been associated with molar incisor hypomineralization. This study aimed to determine if amoxicillin disturbs the enamel mineralization in in vivo experiments. Fifteen pregnant rats were randomly assigned into three groups to received daily phosphatase-buffered saline or amoxicillin as either 100 or 500 mg/kg. Mice received treatment from day 13 of pregnancy to day 40 postnatal. After birth, the offsprings from each litter continued to receive the same treatment according to their respective group. Calcium (Ca) and phosphorus (P) content in the dental hard tissues were analyzed from 60 upper first molars and 60 upper incisors by the complexometric titration method and colorimetric analysis using a spectrophotometer at 680 nm, respectively. Lower incisors were analyzed by X-ray microtomography, it was measured the electron density of lingual and buccal enamel, and the enamel and dentin thickness. Differences in Ca and P content and electron density among the groups were analyzed by one-way ANOVA. There was no significant difference on enamel electron density and thickness among the groups (p > 0.05). However, in incisors, the higher dose of amoxicillin decreased markedly the electron density in some rats. There were no statistically significant differences in Ca (p = 0.180) or P content (p = 0.054), although the higher dose of amoxicillin could affect the enamel in some animals. The amoxicillin did not significantly alter the enamel mineralization and thickness in rats.

Molecular and clinical analyses of the gene encoding the collagen-binding adhesin of Streptococcus mutans.

Streptococcus mutans is a known pathogen of dental caries and its major cell surface antigens have been widely investigated. Recently, an approximately 120 kDa Cnm protein with binding properties to type I collagen was identified, and its encoding gene (cnm) cloned and sequenced. In the present study, we sequenced cnm from 47 different clinical S. mutans strains and found that the nucleotide alignment of the collagen-binding domain was well conserved. We devised a PCR method for identifying the cnm gene, examined the prevalence of cnm-positive S. mutans strains in various mother-child groups, and assessed the significance of such strains for transmission and dental caries. The detection rate of cnm-positive strains was significantly lower in strains isolated from Japanese children in the 2000s (8.0 %) as compared to those isolated in the 1980s (15.8 %) (P<0.05). Furthermore, the presence of S. mutans possessing cnm in salivary specimens collected from 55 S. mutans-positive mother-child pairs was 40 and 32.7 % in the mothers and children, respectively. The frequency of cnm-positive children whose mothers were also positive was 72 %, which was significantly higher than that of cnm-positive children with negative mothers (P<0.0001, odds ratio 17.5). In addition, clinical parameters indicating dental caries were significantly increased in children with cnm-positive S. mutans in saliva (n=13), as compared to those with cnm-negative S. mutans (n=15) and S. mutans-negative children (n=20) (P<0.01). These results indicate that cnm-positive S. mutans strains are closely correlated with dental caries, while vertical transmission in cnm-positive mother-child pairs was also demonstrated.

The Association between Molar-Incisor Hypomineralization and Dental Caries with Socioeconomic Status as an Explanatory Variable in a Group of Finnish Children.

The aim of this study was to investigate if a developmental enamel defect known as Molar-Incisor Hypomineralization (MIH) is associated with dental caries. Socioeconomic status (SES) was examined as a confounding factor between caries and MIH. In this cross-sectional study, 636 children, aged 8 to 13 years, from three towns (two rural areas and one urban area) in Finland were examined for MIH in line with the criteria of the European Academy of Paediatric Dentistry. Caries status for permanent teeth was recorded as decayed, missing and filled teeth (DMFT). Caries experience (DMFT > 0) in the first permanent molars (FPMs) was set as an outcome. SES was determined using a questionnaire completed by parents. The prevalence of MIH was 18.1%. The mean DMFT in FPMs for children with MIH was higher than for their peers, 1.03 ± 1.25 vs. 0.32 ± 0.80 (p = 0.000, Mann-Whitney U test). In a multivariate analysis using the generalized linear mixed model where locality, SES, age and MIH were taken into account as caries risk indicators, MIH was the strongest risk indicator of caries in FPMs (Odds Ratio: 6.60, 95% Confidence Interval: 3.83⁻11.39, p = 0.000). According to the study results, children with MIH have a higher risk for dental caries than children without MIH.

7,12-dimethylbenz[a]anthracene interferes with the development of cultured mouse mandibular molars.

Clinical studies suggest that maternal smoking during pregnancy can reduce the crown size of the child's teeth. Delayed dental age compared with chronological age has also been reported in children whose parents smoke. Among the main components of tobacco smoke are nonhalogenated polycyclic aromatic hydrocarbons (PAHs), many of which are highly toxic. Humans are exposed to PAH compounds mainly via tobacco smoke and diet. The aim of our study was to investigate the effect of PAHs on tooth formation and the function of tooth-forming cells. We exposed mouse (NMRI) E18 mandibular first and second molar explants to 7,12-dimethylbenz[a]anthracene (DMBA), a toxic PAH compound, in organ culture for 7 or 12 days. DMBA concentrations used were 0.1, 0.5, 1, and 2 microM. The mesiodistal width of each first molar (12-day culture) was measured in stereomicroscopic images, and the teeth were analysed histologically. DMBA exposure significantly reduced the mesiodistal width of the first molars. DMBA impaired or delayed amelogenesis and dentinogenesis in both molars at the lowest concentration of 0.1 microM. DMBA affected enamel formation more severely than dentin formation and occasionally prevented amelogenesis completely. Elongation and polarization of ameloblasts were impaired, and blood vessel architecture of the dental papilla (future pulp) was altered. Cusps were thin and sharp. In line with the finding that maternal smoking during pregnancy has an adverse effect on child's tooth development, this study shows the toxic influence of PAHs on tooth development in vitro.

The effect of perinatal TCDD exposure on caries susceptibility in rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the model compound of polychlorinated dibenzo-p-dioxins and furans, is a potent toxicant with the ability to hamper development. Accidental exposure to TCDD has been linked with various developmental dental aberrations in humans, and experimentally it has been shown that TCDD causes, among other defects, hypomineralization of dental hard tissues in rodents. Here, we studied the effect of very low perinatal TCDD exposure on dental caries susceptibility and mineral composition of tooth enamel in rats. Pregnant line C rats (rat line developed in our laboratory) were dosed 0.03-1.0 microg/kg TCDD on gestation day 15 and allowed to give birth and nurse until weaning on postnatal day 21. The offspring were challenged with cariogenic treatment including sugar-rich diet and three inoculations with Streptococcus mutans. Control groups involved animals with or without cariogenic challenge or TCDD treatment. The number of caries lesions in left lower molars was determined by Schiff's staining after 8 weeks of weaning. TCDD treatment increased cariogenic lesions in the enamel at the lowest maternal dose used, 0.03 microg/kg, and at the highest maternal dose, 1 microg/kg, the lesions extended through the enamel to dentin more frequently. Changes in mineral composition measured by electron probe microanalyzer, scanning electron microscopy, and energy-dispersive spectrometry could not explain the increased caries susceptibility. In conclusion, perinatal TCDD exposure can render rat molars more susceptible to caries.

Developmental dental toxicity of dioxin and related compounds--a review.

Non-halogenated polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs, or dioxins), and polychlorinated biphenyls (PCBs) are wide-spread environmental pollutants that have unequivocal adverse effects on different species, including humans. Accidental exposure of children to high amounts of PCDD/Fs has been found to be associated with developmental enamel defects and missing permanent teeth. An association between dioxin exposure via mother's milk and developmental mineralisation defects in permanent first molars was also found in otherwise healthy Finnish children born in the late 1980s but not in those born in the late 1990s. Results of experimental animal studies in vivo and in vitro are compatible with findings in human teeth. In addition to the dose, dental effects of the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), essentially depend on the stage of tooth development at the time of exposure. Accordingly, TCDD arrests early rat and mouse molar tooth development and in more advanced teeth it interferes with mineralisation of enamel and dentine and arrests root development. Expression of the specific dioxin receptor (AhR) in dental cells at TCDD-sensitive stages of tooth development suggests that the dental, like other developmental effects of TCDD, are mediated by the AhR. Early effects also depend on the epidermal growth factor receptor and involve enhanced apoptosis. The lowest TCDD dose (30ng/kg) causing adverse dental effects in rats has been estimated to result in maternal tissue levels approaching the high end of human background range and human milk PCDD/F levels that were associated with enamel defects in children. However, because of the uniform and clear decline in background dioxin and PCB levels in mother's milk during the last twenty years, dioxins are currently likely to be of small or no account as regards developmental dental defects in children. Even so, this is not the case after heavy exposure and little is known about the possible synergistic effects of these toxicants with other chemicals interfering with tooth development.

Expression of the mediators of dioxin toxicity, aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT), is developmentally regulated in mouse teeth.

Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chain. Besides being acutely lethal, the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to many animal species. We have previously found that developing teeth of children may be sensitive to environmental dioxins via their mother's milk and that rat and mouse teeth are dioxin-sensitive throughout their development. The aryl hydrocarbon receptor (AHR) together with the AHR nuclear translocator (ARNT) protein is believed to mediate the toxic effects of dioxins. To study the potential involvement of the AHR-ARNT pathway in the dental toxicity of TCDD, we analysed the expression of AHR and ARNT by in situ hybridization and immunohistochemistry in developing mouse teeth. AHR mRNA first appeared in the epithelium of E12 first molar tooth buds and both proteins were weakly expressed in the bud. After cytodifferentiation the expression was up regulated and became intense in secretory odontoblasts and ameloblasts. The coexpression of AHR and ARNT during early tooth development as well as during the information and mineralization of the dental matrices is suggestive of the AHR-ARNT pathway as a mediator of dental toxicity of TCDD.

The effect of amoxicillin on dental enamel development in vivo

Abstract The exposure to amoxicillin has been associated with molar incisor hypomineralization. This study aimed to determine if amoxicillin disturbs the enamel mineralization in in vivo experiments. Fifteen pregnant rats were randomly assigned into three groups to received daily phosphatase-buffered saline or amoxicillin as either 100 or 500 mg/kg. Mice received treatment from day 13 of pregnancy to day 40 postnatal. After birth, the offsprings from each litter continued to receive the same treatment according to their respective group. Calcium (Ca) and phosphorus (P) content in the dental hard tissues were analyzed from 60 upper first molars and 60 upper incisors by the complexometric titration method and colorimetric analysis using a spectrophotometer at 680 nm, respectively. Lower incisors were analyzed by X-ray microtomography, it was measured the electron density of lingual and buccal enamel, and the enamel and dentin thickness. Differences in Ca and P content and electron density among the groups were analyzed by one-way ANOVA. There was no significant difference on enamel electron density and thickness among the groups (p > 0.05). However, in incisors, the higher dose of amoxicillin decreased markedly the electron density in some rats. There were no statistically significant differences in Ca (p = 0.180) or P content (p = 0.054), although the higher dose of amoxicillin could affect the enamel in some animals. The amoxicillin did not significantly alter the enamel mineralization and thickness in rats.

Developmental dental aberrations after the dioxin accident in Seveso.

Children's developing teeth may be sensitive to environmental dioxins, and in animal studies developing teeth are one of the most sensitive targets of toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Twenty-five years after the dioxin accident in Seveso, Italy, 48 subjects from the contaminated areas (zones A and B) and in patches lightly contaminated (zone R) were recruited for the examination of dental and oral aberrations. Subjects were randomly invited from those exposed in their childhood and for whom frozen serum samples were available. The subjects were frequency matched with 65 subjects from the surrounding non-ABR zone for age, sex, and education. Concentrations of TCDD in previously analyzed plasma samples (zone ABR subjects only) ranged from 23 to 26,000 ng/kg in serum lipid. Ninety-three percent (25 of 27) of the subjects who had developmental enamel defects had been < 5 years of age at the time of the accident. The prevalence of defects in this age group was 42% (15 of 36) in zone ABR subjects and 26% (10 of 39) in zone non-ABR subjects, correlating with serum TCDD levels (p = 0.016). Hypodontia was seen in 12.5% (6 of 48) and 4.6% (3 of 65) of the zone ABR and non-ABR subjects, respectively, also correlating with serum TCDD level (p = 0.05). In conclusion, developmental dental aberrations were associated with childhood exposure to TCDD. In contrast, dental caries and periodontal disease, both infectious in nature, and oral pigmentation and salivary flow rate were not related to the exposure. The results support our hypothesis that dioxins can interfere with human organogenesis.

Response of the incisor tooth to 2,3,7,8-tetrachlorodibenzo-p-dioxin in a dioxin-resistant and a dioxin-sensitive rat strain.

Dioxins are ubiquitous environmental pollutants that afflict developing teeth. To find out if the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the continuously erupting rat incisor is associated with the sensitivity to TCDD acute lethality and to see the histological basis for any macroscopic findings, we exposed 25 resistant Han/Wistar (Kuopio; H/W) and 20 sensitive Long-Evans (Turku/AB; L-E) female rats to total doses of 0.17, 1.7, 17, and 170 (only H/W rats) micro g/kg TCDD. Each dose group comprised five animals. The treatment was started when the rats were 10 weeks old and continued for 20 weeks. The exposure time covered two life cycles of the incisor. Stereomicroscopic examination of the dissected mandibles showed color defects and pulpal perforation of the lower incisors at 17 and 170 micro g/kg TCDD. Tissue sections revealed odontoblastic and pulpal cell death and the consequent arrest of dentin formation at the incisal tooth end at the same doses. H/W rat incisors were affected closer to the germinative tooth end at 170 than at 17 micro g/kg TCDD, resulting in a larger perforation. In accordance with the enamel discoloration, the postsecretory enamel organ underwent, albeit inconsistently, precocious squamous metaplasia with pronounced proliferation. Thus, both the mesenchymal and, to a lesser extent, epithelial elements of the forming tooth were affected dose-dependently at relatively high doses of TCDD. Similar responses in both strains implied that the impaired formation of the incisor tooth, at least of its mesenchymal elements, is not associated with the differential resistance of H/W and L-E rats to TCDD acute lethality.

The effect of high temperature on the development of mouse dental enamel in vitro.

OBJECTIVE: Our aim was to study the effect of high temperature (39°C) corresponding to fever on the development of enamel in cultured mouse molars. DESIGN: For morphological studies mandibular molar blocks from E18 mice were cultured for 11 days. After three days at 37°C the teeth were exposed to 39°C for three or five days and returned to 37°C. At the end of culture, the tooth explants were photographed. The heights of the enamel and the crown of the first molars were measured and the enamel/crown height ratio was calculated. The ratios were compared between the groups using the Mann-Whitney test. To analyze gene expression in ameloblasts and odontoblasts with RT-qPCR and in situ hybridization, part of the test explants were cultured for three days at 37°C and then five days at 39°C. Control explants were kept at 37°C for 11 or eight days. RESULTS: The enamel/crown height ratio of the first molars cultured for five days at 39°C was smaller than that of the unexposed (P<0.001). Tooth morphology did not differ between controls and exposed teeth. In qPCR-analysis, dentine sialophosphoprotein showed clearly decreased expression. In situ hybridization showed no dentine sialophosphoprotein expression in preameloblasts. The expressions of bone morphogenic protein 4, matrix metalloproteinase 20, dentine matrix protein 1, amelogenin and osteocalcin showed a trend of downregulation. CONCLUSIONS: High culturing temperature interferes with enamel formation of mouse molars and alters the expression of some genes essential for normal enamel development.

Candidate gene analysis of tooth agenesis identifies novel mutations in six genes and suggests significant role for WNT and EDA signaling and allele combinations.

Failure to develop complete dentition, tooth agenesis, is a common developmental anomaly manifested most often as isolated but also as associated with many developmental syndromes. It typically affects third molars or one or few other permanent teeth but severe agenesis is also relatively prevalent. Here we report mutational analyses of seven candidate genes in a cohort of 127 probands with non-syndromic tooth agenesis. 82 lacked more than five permanent teeth excluding third molars, called as oligodontia. We identified 28 mutations, 17 of which were novel. Together with our previous reports, we have identified two mutations in MSX1, AXIN2 and EDARADD, five in PAX9, four in EDA and EDAR, and nine in WNT10A. They were observed in 58 probands (44%), with a mean number of missing teeth of 11.7 (range 4 to 34). Almost all of these probands had severe agenesis. Only few of the probands but several relatives with heterozygous genotypes of WNT10A or EDAR conformed to the common type of non-syndromic tooth agenesis, incisor-premolar hypodontia. Mutations in MSX1 and PAX9 affected predominantly posterior teeth, whereas both deciduous and permanent incisors were especially sensitive to mutations in EDA and EDAR. Many mutations in EDAR, EDARADD and WNT10A were present in several families. Biallelic or heterozygous genotypes of WNT10A were observed in 32 and hemizygous or heterozygous genotypes of EDA, EDAR or EDARADD in 22 probands. An EDARADD variant were in seven probands present together with variants in EDAR or WNT10A, suggesting combined phenotypic effects of alleles in distinct genes.