Isabel Maria Porto

Enamel mineralization in the absence of maturation stage ameloblasts

The role of maturation stage ameloblasts is not clear yet. The aim of this study was to verify to which extent enamel mineralizes in the absence of these cells. Maturation stage ameloblasts and adjacent dental follicle cells from rat lower incisors were surgically removed and the limits of this removal were marked by notches made in the enamel. Histological analysis confirmed that the ameloblasts had been removed within the limits of the notches. The teeth erupted and when the notches appeared in the mouth, the enamel in the experimental teeth was hard but whitish compared to the yellowish colour of the contralateral incisors used as control. SEM images revealed similar enamel rod arrangement in both groups. Decreased mineral content was observed in some specimens by polarized light microscopy, and microhardness values were much lower in the experimental teeth. FTIR analysis showed that higher amounts of protein were found in most experimental teeth, compared with the control teeth. Enamel proteins could not be resolved on 15% SDS-PAGE gels, suggesting that most of them were below 5kDa. These results suggest that the enamel matured in the absence of ameloblasts has increased protein content and a much lower mineral content, suggesting that maturation stage ameloblasts are essential for proper enamel mineralization.

Exposure to lead exacerbates dental fluorosis

AIM Our aim was to test the hypothesis that co-exposure to lead and fluoride alter the severity of enamel fluorosis. MATERIALS AND METHODS Wistar rats were allocated in four groups: control, and 3 groups that received water containing 100 ppm of fluoride (F), 30 ppm of lead (Pb), or 100 ppm of F and 30 ppm of Pb (F+Pb) from the beginning of gestation. Enamel analysis and F and Pb determinations in enamel, dentine, and bone were performed in 81-day-old animals. Fluorosis was quantified using a new fluorosis index based on the identification of incisor enamel defects (white bands and white islets, representing hypomineralization, and cavities) weighted according to their severity and quantity. Hypomineralization was validated histopathologically by polarizing microscopy and microradiography. Scores were given by two blinded calibrated examiners (intra and interexaminer kappa values were 0.8 and 0.86, respectively). RESULTS The control and the Pb groups presented normal enamel. The F+Pb group presented more severe enamel defects compared with the F group (P<0.0001). CONCLUSIONS This study shows that lead exacerbates dental fluorosis in rodents, suggesting that co-exposure to lead may affect the degree of fluorosis.

Recovery and identification of mature enamel proteins in ancient teeth.

Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science.

Morphological characterization of rat incisor fluorotic lesions

The morphological characterization of fluorotic rat incisor enamel was carried out. Experimental adult animals received drinking water with 45 mg F/L of fluoride, and the control group received distilled water. Fluoride concentrations found in the control and fluorosis groups were 0.04 and 0.09 microg/mL (plasma), 0.26 and 0.66 microg/mg (whole tibia), and 0.24 and 2.3 microg/mg (tibia surface), with P < or = 0.001 for all comparisons between the groups. A succession of white and pigmented bands was observed in the fluorotic rat incisors. Under polarizing light microscopy, cross-sections of superficial areas corresponding to the white bands (from the surface to approximately 20 microm) showed high positive birefringence. These fluorotic lesions also exhibited the lowest resistance to superficial acid etching. No morphological differences in inner enamel were seen under scanning electron microscopy. In fluorotic enamel, only the surface layer related to the white areas presented lower birefringence compared with the enamel of control teeth and the surface layer of the pigmented areas (normal ones) of fluorotic teeth. In conclusion, the white bands of fluorotic rat enamel represent hypomineralized superficial areas and are not subsurface lesions. The detailed description of these lesions is important to understand dental fluorosis.

In Situ Zymography and Immunolabeling in Fixed and Decalcified Craniofacial Tissues

In situ zymography is a very important technique that shows the proteolytic activity in sections and allows researchers to observe the specific sites of proteolysis in tissues or cells. It is normally performed in non-fixed frozen sections and is not routinely performed in calcified tissues. In this study, we describe a technique that maintains proteolytic activity in fixed and decalcified sections obtained after routine paraffin sectioning in conventional microtome and cryostat sections. We used adult rat hemimandibles, which presented bone, enamel, and dentine matrices; the substrate used was dye-quenched-gelatin. Gelatinolytic activity was colocalized with MMP-2 using fluorescent antibodies. Specific proteolytic activity was observed in all sections, compatible with metalloproteinase activity, particularly in dentine and bone. Furthermore, matrix metalloproteinase-2 was colocalized to the sites of green fluorescence in dentine. In conclusion, the technique presented here will allow in situ zymography reactions in fixed, decalcified, and paraffin-embedded tissues, and we showed that paraformaldehyde-lysine-periodate–fixed cryostat sections are suitable for colocalization of gelatinolytic activity and protein labeling with antibodies. (J Histochem Cytochem 57:615–622, 2009)

Organic and inorganic content of fluorotic rat incisors measured by FTIR spectroscopy

Details on how fluoride interferes in enamel mineralization are still controversial. Therefore, this study aimed at analyzing the organic contents of fluorosis-affected teeth using Fourier Transformation Infrared spectroscopy. To this end, 10 male Wistar rats were divided into two groups: one received 45 ppm fluoride in distilled water for 60 days; the other received distilled water only. Then, the lower incisors were removed and prepared for analysis by two FTIR techniques namely, transmission and micro-ATR. For the first technique, the enamel was powdered, whereas in the second case one fluorotic incisor was cut longitudinally for micro-ATR. Using transmission and powdered samples, FTIR showed a higher C-H content in the fluorotic enamel compared with control enamel (p<0.05, n=4 in the flurotic, and n=5 in the control group). Results from the micro-ATR-FTIR spectroscopic analysis on one longitudinally cut incisor carried out at six points reveal a higher C-H bond content at the surface of the enamel, with values decreasing toward the dentine-enamel junction, and reaching the lowest values at the subsuperficial enamel. These results agree with the morphological data, which indicate that in the rat incisor the fluorotic lesion is superficial, rather than subsuperficial, as in the case of human enamel. The results also suggest that the increased C-H bond content may extend toward the more basal enamel (intraosseous), indicating that fluorotic enamel may intrinsically contain more protein. Finally, particularly when coupled to ATR, FTIR is a suitable tool to study the rat incisor enamel, which is a largely used model of normal and abnormal amelogenesis. Further studies along this line may definitely answer some questions regarding protein content in fluorotic enamel as well as their origin.