Effects of Pamidronate on Dental Enamel Formation Assessed by Light Microscopy, Energy-Dispersive X-Ray Analysis, Scanning Electron Microscopy, and Microhardness Testing.

The aim of the present work was to investigate birefringence and morphology of the secretory-stage enamel organic extracellular matrix (EOECM), and structural and mechanical properties of mature enamel of upper incisors from adult rats that had been treated with pamidronate disodium (0.5 mg/kg/week for 56 days), using transmitted polarizing and bright-field light microscopies (TPLM and BFLM), energy-dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM) and microhardness testing. BFLM showed no morphological changes of the EOECM in pamidronate and control groups, but TPLM revealed a statistically significant reduction in optical retardation values of birefringence brightness of pamidronate-treated rats when compared with control animals (p0.05). The present study indicates that pamidronate can affect birefringence of the secretory-stage EOECM, which does not seem to be associated with significant changes in morphological and/or mechanical properties of mature enamel.

Fluoride effect on the secretory-stage enamel organic extracellular matrix of mice.

The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.

Birefringence of the secretory-stage enamel organic extracellular matrix from rats submitted to successive injections of bisphosphonates.

The aim of the present study was to assess birefringence of the secretory-stage enamel organic extracellular matrix (ECM) and mechanical properties of mature enamel from rats treated with bisphosphonates. Longitudinal sections were obtained from upper incisors of rats that had been submitted to injections of bisodic etidronate (8 mg/Kg/day), sodium alendronate (30 microg/Kg/day), or sodium chloride as control (8 mg/Kg/day) for 42 days. Sections were immersed in 80% glycerin for 30 min and optical retardation of birefringence brightness in the secretory-stage enamel organic ECM was determined in nanometers. Etidronate-treated rats exhibited extensive morphological changes in the secretory-stage enamel organic ECM inclusive nonbirefringent conspicuous incremental lines, but presented optical retardation values similar to those showed by control rats (p > 0.05). Birefringence of secretory enamel organic ECM from etidronate rats presented an irregular aspect. Alendronate-treated rats did not show morphological alterations in the secretory-stage enamel organic ECM, however, they presented significant reduction in optical retardation of birefringence brightness when compared with control and etidronate rats (p < 0.01). Alendronate and etidronate groups exhibited reductions of approximately 6-10% in mature enamel cross-sectional microhardness when compared with control group (p < 0.01). Scanning electron microscopy analysis showed extensive alterations in mature enamel only from etidronate group with absence of enamel rods. The present work shows that bisphosphonates can affect the birefringence of the secretory-stage enamel organic ECM. The results presented here suggest that alterations in the supramolecular organization of the secretory-stage enamel organic ECM are a plausible mechanism by which environmental factors may cause enamel defects.

Amelogenin- and enamelysin (Mmp-20)-deficient mice display altered birefringence in the secretory-stage enamel organic extracellular matrix.

Dental enamel is the most mineralized tissue of vertebrate organisms. Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. The formation of an ordered enamel organic extracellular matrix (ECM) seems be a crucial step for the proper formation of mineral phase. Polarizing microscopy demonstrates that the ordered supramolecular structure of the secretory-stage enamel organic ECM is strongly birefringent. In the present work we analyzed the birefringence of secretory-stage enamel organic ECM in amelogenin (Amelx)- and enamelysin (Mmp20)-deficient mice. Female Amelx+/- animals showed significant reduction in optical retardation values when compared with the Amelx+/+ subgroup (p=0.0029). The secretory-stage enamel organic ECM of the Amelx-/- subgroup did not exhibit birefringence. The secretory-stage enamel organic ECM of Mmp20-/- mice showed a significant decrease in optical retardation as compared with Mmp20+/+ and Mmp20+/- mice (p=0.0000). Mmp20+/- and Mmp20+/+ mice exhibited similar birefringence (p=1.0000). The results presented here support growing evidence for the idea that the birefringence of secretory-stage enamel organic ECM is influenced by the ordered supramolecular organization of its components.

Management of ameloblastic fibro-odontoma in a 6-year-old girl preserving the associated impacted permanent tooth.

The ameloblastic fibro-odontoma (AFO) is a rare odontogenic tumor that occurs predominantly in children and is generally associated with unerupted teeth. The choice of treatment for this entity is conservative surgery with enucleation and its prognosis is excellent. However, preserving the associated impacted teeth may make complete removal of the lesion difficult and may explain some cases of recurrence. A case of AFO in a 6-year-old girl treated with enucleation and preservation of an impacted lower left first permanent molar is reported. After two years of follow-up, there were no signs of recurrence and complete spontaneous eruption of the preserved tooth was observed. The present report indicates that the degree of involvement of the impacted tooth by AFO is a fundamental aspect to be considered during radiological analysis and surgical assessment. We concluded that preservation of the impacted permanent teeth associated with this tumor should always be considered and carried out with caution, since they are clearly not included in the surgical cavity.

Anisotropic properties of the enamel organic extracellular matrix.

Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. This supramolecular ensemble controls the nucleation of the crystalline mineral phase. The detection of anisotropic properties by polarizing microscopy has been extensively used to detect macromolecular organizations in ordinary histological sections. The aim of this work was to study the birefringence of enamel organic matrix during the development of rat molar and incisor teeth. Incisor and molar teeth of rats were fixed in 2% paraformaldehyde/0.5% glutaraldehyde in 0.2 M phosphate-buffered saline (PBS), pH 7.2, and decalcified in 5% nitric acid/4% formaldehyde. After paraffin embedding, 5-microm-thick sections were obtained, treated with xylene, and hydrated. Form birefringence curves were obtained after measuring optical retardations in imbibing media, with different refractive indices. Our observations showed that enamel organic matrix of rat incisor and molar teeth is strongly birefringent, presenting an ordered supramolecular structure. The birefringence starts during the early secretion phase and disappears at the maturation phase. The analysis of enamel organic matrix birefringence may be used to detect the effects of genetic and environmental factors on the supramolecular orientation of enamel matrix and their effects on the structure of mature enamel.

Association between PAX-9 promoter polymorphisms and hypodontia in humans.

Hypodontia, the congenital absence of one or a few teeth, is one of the most common alterations of the human dentition. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Hypodontia does not represent a serious public health problem, but it may cause masticatory and speech dysfunctions, and esthetic problems. PAX 9 is believed to play an important role in tooth development. It is expressed at initiation, bud, cap, and bell stages of odontogenesis. Mutations in PAX 9 coding sequences have been implicated in autosomal dominant oligodontia affecting predominantly permanent molars and second premolars. Here, we report two polymorphisms in the promoter region of PAX 9 gene that are associated with hypodontia. DNA was extracted from buccal epithelial cells of 106 healthy Control individuals and of 102 unrelated individuals with hypodontia. PCR-RFLP was employed in the investigation of G-1031 A and T-912 C polymorphisms. Significant differences were obtained comparing Control and Test groups. Alleles G and T were found at a significant higher frequency in individuals with hypodontia, whereas alleles A and C were more frequent in Control subjects, p=0.0094 and 0.0086, respectively. The GT haplotype was significantly more prevalent in the hypodontia group, while the AC haplotype was more frequent in the Control group. These results indicate that polymorphisms in the promoter region of PAX 9 gene may have an influence on the transcriptional activity of this gene and are associated with hypodontia in humans.